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A Comparison of Mesenchymal Precursor Cells and Amnion Epithelial Cells for Enhancing Cervical Interbody Fusion in an Ovine Model

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... Ten studies used ovine as large animal models (Table 3) [51][52][53][54][55][56][57]59] while two used swine [50,58]. These studies carried out four-level, three-level, twolevel, or single level spinal fusions surgery all instrumented with screws and bars with the exception of Gupta [57]. ...
... et al. that used a single level noninstrumented lumbar fusion[59]. In these studies both autologous bone marrow or expanded MSCs[50,51,54,55,58] and allogeneic bone marrow or mesenchymal precursor cells[52,53,56,57,59] were used to enhance spinal fusion and all of them involved seeding cells into allografts[50], collagen scaffolds[55], ceramics[51-54, 56, 57, 59], and composites[58] scaffolds. Except for Schubert et al. who use MSCs derived from adipose tissue[50] and Goldschlager et al. that used amnion epithelial cells[57] all the other authors used differentiated MSCs and mesenchymal precursor cells derived from bone marrow but also bone marrow in toto loaded on the scaffolds at different dosages. ...
... Except for Schubert et al. who use MSCs derived from adipose tissue[50] and Goldschlager et al. that used amnion epithelial cells[57] all the other authors used differentiated MSCs and mesenchymal precursor cells derived from bone marrow but also bone marrow in toto loaded on the scaffolds at different dosages. Wheeler et al. also compared different dosages of MSCs[52,53,56]. All the researchers used autografts and grafts without cells as controls and the majority highlighted superior result of the graft associated with MSCs and bone marrow in comparison to the graft alone, while similar[50] or best[51- 53, 55, 56, 59] results were seen for autografts in comparison to grafts associated with MSCs. In addition, Goldschlager et al. showed superior results of mesenchymal precursor cells loaded on Mastergraft material also in comparison to amnion epithelial cells ...
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The use of spinal fusion procedures has rapidly augmented over the last decades and although autogenous bone graft is the “gold standard” for these procedures, alternatives to its use have been investigated over many years. A number of emerging strategies as well as tissue engineering with mesenchymal stem cells (MSCs) have been planned to enhance spinal fusion rate. This descriptive systematic literature review summarizes the in vivo studies, dealing with the use of MSCs in spinal arthrodesis surgery and the state of the art in clinical applications. The review has yielded promising evidence supporting the use of MSCs as a cell-based therapy in spinal fusion procedures, thus representing a suitable biological approach able to reduce the high cost of osteoinductive factors as well as the high dose needed to induce bone formation. Nevertheless, despite the fact that MSCs therapy is an interesting and important opportunity of research, in this review it was detected that there are still doubts about the optimal cell concentration and delivery method as well as the ideal implantation techniques and the type of scaffolds for cell delivery. Thus, further inquiry is necessary to carefully evaluate the clinical safety and efficacy of MSCs use in spine fusion.
... Allogeneic transplantation has the advantage over autologous transplantation in offering patients a single procedure without the need for cell harvesting and expansion, thus providing potent cells in a cost-effective manner to a large population. Other types of stem cells, such as human amnion epithelial cells (hAECs) derived from amniotic tissue, and stem cells obtained from human umbilical cord blood or tissue, are also being used xenogeneically in preclinical studies of the treatment of lumbar spinal degenerative conditions [5, 53]. The ready availability of such cells, derived from discarded pregnancy tissues and thus without ethical constraints, and the development of large cell banks to store such cells may make them a convenient source of stem cells for use in degenerative spinal applications, should preclinical efficacy be demonstrated . ...
... Our group has evaluated the ability of hAECs to enhance cervical interbody fusion in an ovine model. However, despite the demonstration of their safety when implanted in the cervical spine, they were found to be inferior to MPCs in promoting fusion [53, 55]. To our knowledge, AECs have not yet been used clinically in intervertebral disc cartilage or spinal regenerative studies. ...
Article
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Degenerative conditions of the lumbar spine are extremely common. Ninety percent of people over the age of 60 years have degenerative change on imaging; however, only a small minority of people will require spine surgery (Hicks et al. Spine (Phila Pa 1976) 34(12):1301-1306, 2009). This minority, however, constitutes a core element of spinal surgery practice. Whilst the patient outcomes from spinal surgeries have improved in recent years, some patients will remain with pain and disability despite technically successful surgery. Advances in regenerative medicine and stem cell therapies, particularly the use of mesenchymal stem cells and allogeneic mesenchymal precursor cells, have led to numerous clinical trials utilising these cell-based therapies to treat degenerative spinal conditions. Through cartilage formation and disc regeneration, fusion enhancement or via modification of pain pathways, stem cells are well suited to enhance spinal surgery practice. This review will focus on the outcomes of lumbar spinal procedures and the role of stem cells in the treatment of degenerative lumbar conditions to enhance clinical practice. The current status of clinical trials utilising stem cell therapies will be discussed, providing clinicians with an overview of the various cell-based treatments likely to be available to patients in the near future.
... The sheep is of a roughly similar size to humans and, despite its quadrupedal stature, is exposed to very similar mechanical stresses to the human intervertebral disc [76]. The ovine spine has previously been used to model disc degeneration [62][63][64] and test implant devices and in the preclinical investigation of cellular therapies [23,[77][78][79]. Similar to the goat the sheep is a hardy animal with demonstrated ability to tolerate surgical intervention. ...
... A recent review by Oehme et al. [103] comprehensively details the variety of preclinical and clinical trials of novel cell-based therapies for the treatment of lumbar intervertebral disc degeneration. Animal models used in preclinical trials of novel therapies include rat [47], rabbit [54], canine [56], porcine [61], ovine [23,78,79], and rhesus monkeys [104]. The vast majority of animal models described utilized full thickness annular injury with nuclear involvement to induce disc degeneration. ...
Article
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Lower back pain is the leading cause of disability worldwide. Discogenic pain secondary to intervertebral disc degeneration is a significant cause of low back pain. Disc degeneration is a complex multifactorial process. Animal models are essential to furthering understanding of the degenerative process and testing potential therapies. The adult human lumbar intervertebral disc is characterized by the loss of notochordal cells, relatively large size, essentially avascular nature, and exposure to biomechanical stresses influenced by bipedalism. Animal models are compared with regard to the above characteristics. Numerous methods of inducing disc degeneration are reported. Broadly these can be considered under the categories of spontaneous degeneration, mechanical and structural models. The purpose of such animal models is to further our understanding and, ultimately, improve treatment of disc degeneration. The role of animal models of disc degeneration in translational research leading to clinical trials of novel cellular therapies is explored.
... Seven studies (84 levels) reported imaging and/or histology outcomes for ACDF at 12 weeks postoperative using the following groups: autograft, empty titanium cages, titanium cages filled with autograft, and PEEK cages filled with autograft [23][24][25][26][27][28][29]. One of the studies of ACDF with autograft included fixation with an anterior plate [23]. ...
... Fusion results for the ADCF studies were similar, regardless of the interbody treatment (autograft, empty titanium cages, autograft-filled titanium cages, autograft-filled PEEK cages) or the fusion evaluation tool (radiographs, CTs, histology) [23][24][25][26][27][28][29]. All of the identified studies used a 12 week postoperative time frame and fusion scores consistently indicated little to moderate bone formation with a non-bridging bony response from a single endplate. ...
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Purpose The ovine model is often used to evaluate new spine fusion technologies prior to clinical testing. An important aspect of designing sheep surgery protocols is to select the appropriate postoperative time period for comparing fusion outcomes. Unfortunately, determining the ideal study endpoint is complicated by the fact that prior published studies have not used consistent timeframes. Thus, the primary aim of this study was to provide a reference for investigators as to the expected fusion outcomes of control groups at varying timepoints in sheep spine surgery models. Methods We identified published sheep fusion studies using autograft, interbody cages, and/or instrumentation. Fusion data were extracted, converted to a common scale, and analyzed across studied timepoints. Results Overall, 29 studies of 360 fusion levels were identified: 11 ALIF (158 levels), 3 PLIF/TLIF (28 levels), 8 PLF (90 levels), and 7 ACDF (84 levels). Studied timepoints ranged from 4 to 48 weeks postoperative. In general, fusion rates varied across techniques and instrumentation. The time to reach solid fusion differed by as many as 20 weeks between control groups. Conclusions Recommended timeframes for future studies designed to show either superiority over controls or equivalent outcomes with controls were developed based on aggregate results. Designating ideal study endpoints for sheep fusion models has both ethical implications associated with responsible use of animals in research, and economic implications given the cost of animal research. The current results can guide the development of future research methods and help investigators choose appropriate study timelines for various control groups.
... Amniotic epithelial cells showed similar patterns to AMSCs in the sheep (Gloria et al. 2010;Mauro et al. 2010;Mattioli et al. 2012), but the latter were superior in promoting interbody fusion in induced anterior cervical discectomy after allogenic implantation combined with bone grafts (Goldschlager et al. 2011). AECs phenotype, methylation status, immunomodulatory and stemness properties are affected by gestational age (Barboni et al. 2014). ...
... Although implantation of human AMSCs into animals resulted in successful and persistent engraftment in multiple organs and tissues (Lindenmair et al. 2012), their application in veterinary medicine are still limited (Table 5). In fact, except for a study to enhance cervical interbody fusion in an ovine model (Goldschlager et al. 2011) and histological and functional improvement after transplantation in a porcine model of chronic myocardial ischaemia (Kimura et al. 2012), AMSCs were only applied in equine spontaneous tendon injuries. Allogenic transplantation resulted in a quick reduction in tendon size and ultrasonographic cross-sectional area , and the re-injury rate was lower compared to autologous BMMSCs transplantation (Lange-Consiglio et al. 2013c). ...
Article
Over the past decade, stem cell research has emerged as an area of major interest for its potential in regenerative medicine applications. This is in constant need of new cell sources to conceive regenerative medicine approaches for diseases that are still without therapy. Scientists drew the attention towards alternative sources such as foetal adnexa and fluid, as these sources possess many advantages: first of all, cells can be extracted from discarded foetal material and it is non-invasive and inexpensive for the patient; secondly, abundant stem cells can be obtained; and finally, these stem cell sources are free from ethical considerations. Cells derived from foetal adnexa and fluid preserve some of the characteristics of the primitive embryonic layers from which they originate. Many studies have demonstrated the differentiation potential in vitro and in vivo towards mesenchymal and non-mesenchymal cell types; in addition, the immune-modulatory properties make these cells a good candidate for allo- and xenotransplantation. Naturally occurring diseases in domestic animals can be more ideal as disease model of human genetic and acquired diseases and could help to define the potential therapeutic use efficiency and safety of stem cells therapies. This review offers an update on the state of the art of characterization of domestic animals' MSCs derived from foetal adnexa and fluid and on the latest findings in pre-clinical or clinical setting of the stem cell populations isolated from these sources. © 2015 Blackwell Verlag GmbH.
... 91 Numerous other studies describe successes of stem cell-augmented fusion in animal models. [92][93][94][95][96][97][98] Despite success in animal models, limited studies exist in humans, with most studies utilizing autologous bone marrow aspiration followed by an attempt to concentrate MSCs via centrifugation, expansion, or selective cell retention, known as bone marrow aspirate concentrate (BMAC) (Figure 2). Yousef et al 99 retrospectively found successful fusion in all 21 PLF patients given BMAC in a collagen scaffold. ...
Article
BACKGROUND Osteobiologics are engineered materials that facilitate bone healing and have been increasingly used in spine surgery. Autologous iliac crest bone grafts have been used historically, but morbidity associated with graft harvesting has led surgeons to seek alternative solutions. Allograft bone, biomaterial scaffolds, growth factors, and stem cells have been explored as bone graft substitutes and supplements. OBJECTIVE To review current and emerging osteobiologic technologies. METHODS A literature review of English-language studies was performed in PubMed. Search terms included combinations of “spine,” “fusion,” “osteobiologics,” “autologous,” “allogen(e)ic,” “graft,” “scaffold,” “bone morphogenic protein,” and “stem cells.” RESULTS Evidence supports allograft bone as an autologous bone supplement or replacement in scenarios where minimal autologous bone is available. There are promising data on ceramics and P-15; however, comparative human trials remain scarce. Growth factors, including recombinant human bone morphogenic proteins (rhBMPs) 2 and 7, have been explored in humans after successful animal trials. Evidence continues to support the use of rhBMP-2 in lumbar fusion in patient populations with poor bone quality or revision surgery, while there is limited evidence for rhBMP-7. Stem cells have been incredibly promising in promoting fusion in animal models, but human trials to this point have only involved products with questionable stem cell content, thereby limiting possible conclusions. CONCLUSION Engineered stem cells that overexpress osteoinductive factors are likely the future of spine fusion, but issues with applying viral vector-transduced stem cells in humans have limited progress.
... 11 These 2 cell layers are the sources of human amniotic multipotent epithelial cells (hAECs) and amniotic MSCs (AMSCs), respectively, both of which have stem cell-like properties. 4,6,7,[12][13][14][15] The chorion of term fetal membranes consists of the chorionic mesenchymal stromal layer covering the chorionic trophoblast cell layer, which are both of fetal origin. As the fetus grows, the fetal membranes enlarge such that the chorion pushes into, and subsequently adheres to, the overlying decidua parietalis, which is of maternal origin. ...
Article
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Mesenchymal stromal cells (MSCs) from gestational tissues represent promising cell populations with stem cell-like properties for use in regenerative medicine. Previously, we reported that MSCs in the chorionic villi of the human placenta reside in a vascular niche. However, the niche(s) in which MSCs reside in the fetal membranes, another rich source of MSCs, remains to be determined. The cell surface markers STRO-1 and 3G5 were previously employed to identify niches in a variety of tissues and here we use these markers to report the location of the MSC niche in the human decidua parietalis. The cultured decidua parietalis MSCs (DPMSCs) isolated from the choriodecidua component of the fetal membranes possessed stem cell-like properties such as adherence to plastic, colony forming ability, and multipotent differentiation potential. Fluorescence in situ hybridization analysis showed cultured DPMSCs were of maternal origin. Immunocytochemistry demonstrated that cultured DPMSCs stained positively with stem cell surface markers 3G5, CD105, CD106, STRO-1, CD146, CD49a, and α-SMA but were negative for hematopoietic markers (CD117, CD34) and vascular markers (CD34, von Willebrand factor [vWF]). Immunohistochemistry with antibodies to stem cell surface markers and the endothelial markers on term fetal membranes revealed a vascular niche for DPMSCs, which was confirmed by immunofluorescence analysis. Both STRO-1 and vWF fluorescence signals showed substantial overlap, while CD146 and vWF signals showed partial overlap. These observations were consistent with a vascular niche.
... The sheep spine shares many similarities, both anatomically and biochemically, to the human spine, making it increasingly popular as a large animal model for preclinical spine surgery studies123. The ovine spine has been used as a model for disc degeneration456789, to test novel implant devices101112, and as a preclinical model for biological therapies such as stem cell treatments or administration of growth factors13141516. Posterior approaches to the lumbar intervertebral discs, commonly used in human surgery, are difficult in the sheep due to the presence of the spinal cord within the lumbar spinal canal and ossification of the posterior longitudinal ligament (PLL). ...
Article
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The sheep is becoming increasingly used as a large animal model for preclinical spine surgery studies. Access to the ovine lumbar intervertebral discs has traditionally been via an anterior or anterolateral approach, which requires larger wound incisions and, at times, significant abdominal retraction. We present a new minimally invasive operative technique for a far-lateral approach to the ovine lumbar spine that allows for smaller incisions, excellent visualisation of intervertebral discs, and minimal abdominal retraction and is well tolerated by animals with minimal morbidity.
... Our group has evaluated the ability of AECs to enhance cervical interbody fusion. While being safe to implant in the cervical spine, our studies found AECs to be inferior to MPCs in promoting fusion [58,59]. To our knowledge AECs have not yet been evaluated for their efficacy in intervertebral disc regenerative therapies. ...
Article
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Spinal surgery involves the bone-cartilage-neural interface. It is a field of surgery that is rapidly changing and evolving; not only through the development of novel techniques, approaches and devices but also through evidence from large clinical trials assessing indications, efficacy and outcomes. The use of biologics in spine surgery has now become widespread. Biologics in the form of autologous or allogeneic stem cells or progenitor cells are not yet in routine clinical use in spine surgery. However it is likely that they will have a significant role in the future, since increasing numbers of preclinical and clinical studies have demonstrated the safety and efficacy of progenitor cells to treat a variety of spinal conditions. Such studies have paved the way to larger clinical trials. Cell therapies encompass a wide range of stem cell and progenitor cell types. Stem cells subtypes differ in their lineage potential often being described as pluripotent or multipotent, some of which have potential application in therapies to treat diseases of the spine having the ability to differentiate into tissues including bone and cartilage and to secrete factors that promote matrix repair and regeneration. Furthermore, studies have shown that some cells, particularly mesenchymal stromal cells, modulate oxidative stress and secrete cytokines and growth factors that have immunomodulatory, antiinflammatory, angiogenic and antiapoptotic effects. It is these combined characteristics that make cell based therapies prime candidates for advancing current techniques in spine surgery and for providing new strategies directed at targeting the underlying causes of spinal diseases and disorders to promote repair and regeneration. This review will explore the characteristics of various stem cells and other progenitor cells derived from different sources. The authors are not suggesting that all these cells are necessarily suitable clinically. The review will thus focus on their application to both current and potentially future areas of spine surgery based on results of the available evidence and clinical trials. This review will not address spinal cord injury.
... Important for transplantation approaches, hAECs have been evaluated in a variety of animal models of human disease without evidence of xenogeneic reactions and have exhibited minimal major histocompatibility complex class I and II expression, supporting the use of AECs xenogeneically [14,15,[31][32][33][34]. Intravenous delivery of fresh, unfractionated hAECs into newborn rats, or their interperitoneal delivery into newborn swine, led to microchimerism in multiple organs and tissues [31]. ...
Article
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The present study investigated the detailed in vitro osteogenic differentiation process and in vivo bone regenerative property of human amniotic epithelial cells (hAECs). The in vitro osteogenic differentiation process of hAECs was evaluated by biochemical staining, real-time polymerase chain reaction, and immunofluorescence. Next, β-tricalcium phosphate (β-TCP) scaffolds alone or loaded with hAECs were implanted into the alveolar defects of rats. Micro-computed tomography evaluation and histologic studies were conducted. Our results validated the in vitro osteogenic capacity of hAECs by upregulation of Runx2, osterix, alkaline phosphatase, collagen I, and osteopontin, with positive biochemical staining for osteoblasts. An epithelial-mesenchymal transformation process might be involved in the osteogenic differentiation of hAECs by increased expression of transforming growth factor-β1. Our data also demonstrated that in vivo implantation of hAECs loaded on β-TCP scaffolds, not only improved bone regeneration by direct participation, but also reduced the early host immune response to the scaffolds. The presented data indicate that hAECs possess proper osteogenic differentiation potential and a modulatory influence on the early tissue remodeling process, making these cells a potential source of progenitor cells for clinical restoration of the alveolar defect.
... 15,16,50 Moreover, the methodology employed to isolate these MPCs has been scaled up, and the cryogenically preserved cellsfrom a single animal or human donor-have been used as an "off-the-shelf" supply of allogeneic MPCs for applications in a variety of surgical procedures. [12][13][14]38 In the present study we used the STRO-3 antibody to purify the MPCs present in the bone marrow of Suffolk-Merino crossbred sheep. These immunoselected STRO-3 + MPCs retained the highest clonogenic capacity of the bone marrow cells and were free of contaminating hematopoietic accessory cells such as monocytes/macrophages, lymphocytes, and erythrocyte progenitors, which can influence the growth and development of MPCs in unfractionated cultures isolated by plastic adherence. ...
Article
Chronic low-back pain of discal origin is linked strongly to disc degeneration. Current nonsurgical treatments are palliative and fail to restore the disc extracellular matrix. In this study the authors examined the capacity of ovine mesenchymal precursor cells (MPCs) to restore the extracellular matrix of degenerate discs in an ovine model. Three adjacent lumbar discs of 24 adult male sheep were injected intradiscally with chondroitinase-ABC (cABC) to initiate disc degeneration. The remaining lumbar discs were used as normal controls. Three months after cABC injection, the L3-4 discs of all animals were injected with either a high dose (4 × 10(6) cells, in 12 sheep) or low dose (0.5 × 10(6) cells, in 12 sheep) of MPCs suspended in hyaluronic acid (HA). The adjacent L4-5 degenerate discs remained untreated; the L5-6 discs were injected with HA only. The animals were euthanized at 3 or 6 months after MPC injections (6 sheep from each group at each time point), and histological sections of the lumbar discs were prepared. Radiographs and MR images were obtained prior to cABC injection (baseline), 3 months after cABC injection (pretreatment), and just prior to necropsy (posttreatment). Injection of cABC decreased the disc height index (DHI) of target discs by 45%-50%, confirming degeneration. Some recovery in DHI was observed 6 months after treatment in all cABC-injected discs, but the DHI increased to within baseline control values only in the MPC-injected discs. This improvement was accompanied by a reduction in MRI degeneration scores. The histopathology scores observed at 3 months posttreatment for the high-dose MPC-injected discs and at 6 months posttreatment for the low-dose MPC-injected discs were significantly different from those of the noninjected and HA-injected discs (p <0.001) but not from the control disc scores. On the basis of the findings of this study, the authors conclude that the injection of MPCs into degenerate intervertebral discs can contribute to the regeneration of a new extracellular matrix.
... Historically, most clinical reports on the use of placental tissues have discussed the use of amniotic membranes for an array of clinical applications since the 1920's [15][16][17][18][19]. These uses include general surgery [20][21], corneal surgery [22][23][24], plastic surgery [25], burns and wound care [26][27][28][29][30][31][32], sports medicine [33][34], foot and ankle procedures [35][36], spine and dura repair [37][38][39][40][41][42][43][44], nerve wrap or dural covering [39,42,45], and tendon repair [46]. ...
Article
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Damaged connective tissue commonly leads to lower extremity injuries. These injuries can result in inflammation, reduced mobility, and chronic pain. Conservative treatment may include orthotics, offloading the injury, physical therapy, and/or NSAIDs. If conservative treatment fails, surgical intervention may be required. Even after successful surgery, these procedures often result in reduced joint mobility and tendon or ligament strength. A novel flowable tissue matrix allograft, derived from human placental connective tissue, has recently been made available for minimally invasive treatment of damaged or inadequate tissue (PX50®, Human Regenerative Technologies LLC, Redondo Beach, CA). Based on the universal role of connective tissue in the body, and its reported antimicrobial, anti-adhesive, and anti-inflammatory properties, we assessed the effects of using this placental tissue matrix in the treatment of a series of lower extremity injuries. In this pilot study, 9 of 10 patients reported pain levels of 2 or less by week four using the VAS pain scale. This short-term pilot study effectively shows that injectable, flowable amniotic allografts can be used for orthopedic sports injuries of the lower extremities.
... [50] BMSCs have been successfully used in clinical applications including spinal fusion, segmental bone defects, and craniotomy defects. [51][52][53] The osteogenic potential of BMSC was found to be higher than that of osteoblasts in a bovine in vitro model. [54] Importantly, BMSCs loaded on scaffolds implanted into an osteochondral defect resulted in both osteogenesis and chondrogenesis. ...
Article
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Skeletal regenerative medicine emerged as a field of investigation to address large osseous deficiencies secondary to congenital, traumatic, and post-oncologic conditions. Although autologous bone grafts have been the gold standard for reconstruction of skeletal defects, donor site morbidity remains a significant limitation. To address these limitations, contemporary bone tissue engineering research aims to target delivery of osteogenic cells and growth factors in a defined three dimensional space using scaffolding material. Using bone as a template, biomimetic strategies in scaffold engineering unite organic and inorganic components in an optimal configuration to both support osteoinduction as well as osteoconduction. This article reviews the various structural and functional considerations behind the development of effective biomimetic scaffolds for osteogenesis and highlights strategies for enhancing osteogenesis.
... These cells show stem cell-like properties. 3,15,16,[23][24][25][26] The chorion membrane consists of the chorionic mesenchymal stromal layer that covers the chorionic trophoblast cell layer. Both cellular layers are of fetal origin. ...
Article
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Mesenchymal stem/multipotent stromal cells (MSCs) from the human placenta show stem cell-like properties useful for regenerative medicine. Previously, we reported that MSCs isolated from the fetal part of human term placentae have characteristics, which make them a potential candidate for regenerative medicine. In this study, we characterized MSC isolated from the maternal part of human term placenta. The MSCs were isolated from the decidua parietalis (DPMSCs) of human placenta using a digestion method and characterized by colony-forming unit assay and the expression of MSC markers by flow cytometry technique. In addition, DPMSC differentiation into the 3 mesenchymal lineages was also performed. Moreover, the gene and protein expression profiles of DPMSCs were identified by real-time polymerase chain reaction and flow cytometry techniques, respectively. Furthermore, proteins secreted by DPMSCs were detected by sandwich enzyme-linked immunosorbent assays. Finally, the proliferation and migration potentials of DPMSCs were also determined. The DPMSCs were positive for MSC markers and negative for hematopoietic and endothelial markers, as well as costimulatory molecules and HLA-DR. Functionally, DPMSCs formed colonies and differentiated into chondrocytes, osteocytes, and adipocytes. In addition, they proliferated and migrated in response to different stimuli. Finally, they expressed and secreted many biological and immunological factors with multiple functions. Here, we carry out an extensive characterization of DPMSCs of human placenta. We report that these cells express and secrete a wide range of molecules with multiple functions, and therefore, we suggest that these cells could be an attractive candidate for cell-based therapy.
Article
Management of degenerative spine pathologies frequently leads to the need for spinal fusion, where bone growth is induced towards stabilization of the interventioned spine. Autologous bone graft (ABG) remains the gold standard inducer, while new bone graft substitutes attempt to achieve effective de novo bone formation and solid fusion. Limited fusion outcomes have driven motivation for more sophisticated and multidisciplinary solutions, involving new biomaterials and/or biologics, through innovative delivery platforms. The present review will analyze the most recent body of literature focused on new approaches for consistent bone fusion of spinal vertebrae, including the development of new biomaterials that pursue physical and chemical aptitudes; the delivery of growth factors (GF) to accelerate new bone formation; and the use of cells to improve functional bone development. Bone graft substitutes currently in clinical practice, such as demineralized bone matrix and ceramics are still used as starting point for study of new bioactive agents. Polyesters such as polycaprolactone and polylactic acid arise as platforms for development of composites, where a mineral element and cell/growth factors constitute the delivery system. Exciting fusion outcomes were obtained in several small and large animal models with these. On what regards bioactive agents, mesenchymal stem cells, preferentially derived from the bone marrow or adipose tissue, were studied in this context. Autologous and allogeneic approaches, as well as osteogenically differentiated cells, have been tested. These cell sources have further been genetically engineered for specific growth factor expression. Nevertheless, results on fusion efficacy with cells have been inconsistent. On the other hand, delivery of growth factors (most commonly BMP-2), have provided favorable outcomes. Complications related to burst release and dosing are still target of research through development of controlled release systems or alternative GF such as NELL-1, Oxysterols or COMP-Ang1. Promising solutions with new biomaterial and GF compositions are becoming closer to the human patient, as these evidence high fusion performance, while offering cost and safety advantages. The use of cells has not yet proven solid benefits, whereas further understanding of cell behavior remains a challenge.
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There are $151,000 lumbar fusions performed annually in the United States, 1 and a large number of these patients suffer from considerable morbidity due to the harvesting of autogenous iliac crest bone graft (ICBG). Fifteen to 60% of patients complain of pain at the donor site 1 to 2 years following surgery. 2–6 Pseudarthrosis rates following ICBG fusion have been reported to occur in as many as 15% of patients, with some requiring subsequent revision surger-ies, with additional associated morbidity. 2 Complication rates due to the iliac crest harvest have varied widely, ranging from 9.4 to 49%. 6 In one prospective study, 6 at 12 months following surgery, 16.5% of patients reported pain that was more severe at the harvest site than the primary surgical site, almost 30% reporting continued numbness, and 15% had some difficulty walking. Although initial studies showed promise that the use of recombinant human bone morphogenetic protein-2 in spine surgery could obviate the need for the ICBG and its associated pain, the recent controversy surrounding its potential risks (e.g., radiculopathy, retrograde ejaculation, etc.) encourages the spine surgery community to consider alternative ap-proaches to enhance spine fusion. Identifying ideal spine fusion extenders (i.e., products that can be used alongside the bone graft) as well as bone graft replacements that can potentially replace ICBG may potentially eliminate the associated morbidity with the current standard procedure. The ideal graft re-placement would mirror iliac crest; that is, it would have osteoconductive, osteogenic, and osteoinductive capacity. Adult mesenchymal stem cells (MSCs) have shown poten-tial for bone regeneration. These cells can be isolated from bone marrow, adipose, and muscle tissue, among others, and may be induced to differentiate into osteogenic cells to enhance spinal fusion. 7 The use of MSCs to promote spinal fusion will be summarized here.
Spinal pathologies are a major burden on society and individuals. Recent years have seen a large number of studies dedicated to the use of stem cells in spinal surgery. This review focuses on recent advances and controversies regarding the applications of stem cells in spinal fusion surgery, spinal cord injury and intervertebral disc degeneration. There are significant concerns regarding the ethics and risks of stem cell use. Animal models do not always accurately depict the human condition. While a great deal has been achieved, successful translation into clinical practice is needed. However there is no doubt that stem cells have a major role to play in the future management of spinal conditions.
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Study Design Narrative literature review. Objectives Placental tissue, amniotic/chorionic membrane, and umbilical cord have seen a recent expansion in their clinical application in various fields of surgery. It is important for practicing surgeons to know the underlying science, especially as it relates to spine surgery, to understand the rationale and clinical indication, if any, for their usage. Methods A literature search was performed using PubMed and MEDLINE databases to identify studies reporting the application of placental tissues as it relates to the practicing spine surgeon. Four areas of interest were identified and a comprehensive review was performed of available literature. Results Clinical application of placental tissue holds promise with regard to treatment of intervertebral disc pathology, preventing epidural fibrosis, spinal dysraphism closure, and spinal cord injury; however, there is an overall paucity of high-quality evidence. As such, evidence-based guidelines for its clinical application are currently unavailable. Conclusions There is no high-level clinical evidence to support the application of placental tissue for spinal surgery, although it does hold promise for several areas of interest for the practicing spine surgeon. High-quality research is needed to define the clinical effectiveness and indications of placental tissue as it relates to spine surgery.
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Study Design Large animal research. Objective Lumbar discectomy is the most commonly performed spinal surgical procedure. We investigated 2 large animal models of lumbar discectomy in order to study the regenerative capacity of mesenchymal stem cells following disc injury. Methods Twelve adult ewes underwent baseline 3-T magnetic resonance imaging (MRI) followed by lumbar intervertebral disc injury by either drill bit (n = 6) or annulotomy and partial nucleotomy (APN) (n = 6). Necropsies were performed 6 months later. Lumbar spines underwent 3-T and 9.4-T MRI prior to histological, morphological and biochemical analysis. Results Drill bit-injured (DBI) and APN-injured discs demonstrated increased Pfirrmann grades relative to uninjured controls (P < .005), with no difference between the 2 models. Disc height index loss was greater in the APN group compared with the DBI group (P < .005). Gross morphology injury scores were higher in APN than DBI discs (P < .05) and both were higher than controls (P < .005). Proteoglycan was reduced in the discs of both injury models relative to controls (P < .005), but lower in the APN group (P < .05). Total collagen of the APN group disc regions was higher than DBI and control discs (P < .05). Histology revealed more matrix degeneration, vascular infiltration, and granulation in the APN model. Conclusion Although both models produced disc degeneration, the APN model better replicated the pathobiology of human discs postdiscectomy. We therefore concluded that the APN model was a more appropriate model for the investigation of the regenerative capacity of mesenchymal stem cells administered postdiscectomy.
Article
Introduction Back pain is a common ailment affecting individuals around the globe. Animal models to understand the back pain mechanism, treatment modalities, and spinal cord injury are widely researched topics worldwide. Despite the presence of several animal models on disc degeneration and Spinal Cord Injury, there is a lack of a comprehensive review. Material and method A methodological narrative literature review was carried out for the study. A total of 1273 publications were found, out of which 763 were related to spine surgery in animals. The literature with full-text availability was selected for the review. Scale for the Assessment of Narrative Review Articles (SANRA) guidelines was used to assess the studies. Only English language publications were included which were listed on PubMed. A total of 113 studies were shortlisted (1976–2019) after internal validation scoring. Result The animal models for spine surgery ranged from rodents to primates. These are used to study the mechanisms of back pain as well as spinal cord injuries. The models could either be created surgically or through various means like use of electric cautery, chemicals or trauma. Genetic spine models have also been documented in which the injuries are created by genetic alterations and knock outs. Though the dorsal approach is the most common, the literature also mentions the anterior and lateral approach for spine surgery animal experiments. Conclusion There are no single perfect animal models to represent and study human models. The selection is based on the application and the methodology. Careful selection is needed to give optimum and appropriate results.
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Bone repair and regeneration is a dynamic process that involves a complex interplay between the (1) ground substance; (2) cells; and (3) milieu. Each constituent is integral to the final product, but it is often helpful to consider each component individually. While bone tissue engineering has capitalized on a number of breakthrough technologies, one of the most valued advancements is the incorporation of mesenchymal stem cells (SCs) into bone tissue engineering applications. With this new idea, however, came new found problems of guiding SC differentiation. Moreover, investigators are still working to understand which SCs source produces optimal bone formation in vitro and in vivo. Bone marrow-derived mesenchymal SCs and adipose-derived SCs have been researched most extensively, but other SC sources, including dental pulp, blood, umbilical cord blood, epithelial cells reprogrammed to become induced pluripotent SCs, among others, are being investigated. In Part II of this review series, we discuss the variety of cell types (e.g., osteocytes, osteoblasts, osteoclasts, chondrocytes, mesenchymal SCs, and vasculogenic cells) important in bone tissue engineering.
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Low back pain is a major health problem impacting millions of people, which the intervertebral disc degeneration is considered as a major cause. In recent years stem cell research has been a great hope for orthopedic regenerative medicine. In vitro experiment, animal research and preclinical studies provided promising results in the treatment of disc regeneration. A few clinical trials also demonstrated the feasibility and efficacy of disc regeneration and structural fusion. The identification of progenitor cells within the intervertebral disc provides another mechanism for the disc degeneration etiology and disc regeneration. This paper aims to provide a better understanding of current basic science research and clinical studies focusing on stem cells in disc degeneration and spine fusion.
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Anterior cervical discectomy with fusion is a common surgical procedure for patients suffering pain and/or neurological deficits and unresponsive to conservative management. For decades, autologous bone grafted from the iliac crest has been used as a substrate for cervical arthrodesis. However patient dissatisfaction with donor site morbidity has led to the search for alternative techniques. We present a literature review examining the progress of available grafting options as assessed in human clinical trials, considering allograft-based, synthetic, factor- and cell-based technologies.
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There is an unmet need for a procedure that could generate a biological disc substitute while at the same time preserving the normal surgical practice of achieving anterior cervical decompression. The objective of the present study was to test the hypothesis that adult allogeneic mesenchymal progenitor cells (MPCs) formulated with a chondrogenic agent could synthesize a cartilaginous matrix when implanted into a biodegradable carrier and cage, and over time, might serve as a dynamic interbody spacer following anterior cervical discectomy (ACD). Eighteen ewes were divided randomly into 3 groups of 6 animals. Each animal was subjected to C3-4 and C4-5 ACD followed by implantation of bioresorbable interbody cages and graft containment plates. The cage was packed with 1 of 3 implants. In Group A, the implant was Gelfoam sponge only. In Group B, the implant consisted of Gelfoam sponge with 1 million MPCs only. In Group C, the implant was Gelfoam sponge with 1 million MPCs formulated with the chondrogenic agent pentosan polysulfate (PPS). In each animal the cartilaginous endplates were retained intact at 1 level, and perforated in a standardized manner at the other level. Allogeneic ovine MPCs were derived from a single batch of immunoselected and culture-expanded MPCs isolated from bone marrow of outbred sheep (mixed stock). Radiological and histological measures were used to assess cartilage formation and the presence or absence of new bone formation. The MPCs with or without PPS were safe and well-tolerated in the ovine cervical spine. There was no significant difference between groups in the radiographic or histological outcome measures, regardless of whether endplates were perforated or retained intact. According to CT scans obtained at 3 months after the operation, new bone formation within the interbody space was observed in the Gelfoam only group (Group A) in 9 (75%) of 12 interbody spaces, and 11 (92%) of 12 animals in the MPC cohort (Group B) had new bone formation within the interbody space. Significantly, in the MPC & PPS group (Group C), there were only 1 (8%) of 12 levels with new bone formation (p = 0.0009 vs Group A; p = 0.0001 vs Group B). According to histological results, there was significantly more cartilaginous tissue within the interbody cages of Group C (MPC & PPS) compared with both the control group (Group A; p = 0.003) and the MPC Group (p = 0.017). This study demonstrated the feasibility of using MPCs in combination with PPS to produce cartilaginous tissue to replace the intervertebral disc following ACD. This biological approach may offer a means preserving spinal motion and offers an alternative to fusion to artificial prostheses.
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Undecalcified bone histology demonstrates the micro-architecture of bone. It shows both the mineralised and cellular components of bone, which provides vital information on bone turnover or bone formation and resorption. This has tremendous importance in a variety of clinical and research applications. It yields beautiful images1 and allows for techniques such as fluorochrome assessment and histomorphometry2. Fluorochrome analysis is a technique where fluorescent dyes that bind to calcium are injected at a particular time point, which allows for quantification of the amount of mineralisation at that given time. Histomorphometry is a process of bone quantification at the microscopic level. Performing undecalcified bone histology is technically challenging, particularly with large size specimens. It requires variations in technique from those used in standard paraffin embedded histology. This video illustrates the process of producing good quality sections and demonstrates the technical difficulties and methods with which to overcome them. Specimen preparation, fixation and processing are achieved with a manner similar to other soft tissues, however due to the density and lower permeability of bone considerably longer fixation and processing times are required, often taking several weeks. Embedding is achieved using a supporting medium with similar or equal hardness and density to the bone such as methacrylate- based resins, but unlike paraffin infiltration and embedding, this is an irreversible step. Sectioning can be achieved by grinding which produces a thicker section, which is optimal for studies such as fluorochrome analysis. This is best achieved using a diamond blade on a macrotome. Alternatively, thinner sections can be produced for light microscopy and this is achieved using a sledge microtome with a very sharp blade. The sledge microtome provides the additional strength and stability required for large, hard blocks. Resin embedded sections can be stained with a variety of stains, which are demonstrated.
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Anterior cervical discectomy and fusion (ACDF) is the most common surgical operation for cervical radiculopathy and/or myelopathy in patients who have failed conservative treatment. Since the operation was first described by Cloward and Smith and Robinson in 1958, a variety refinements in technique, graft material and implants have been made. In particular, there is a need for safe osteoinductive agents that could benefit selected patients. The ovine model has been shown to have anatomical, biomechanical, bone density and radiological properties that are similar to the human counterpart, the most similar level being C3/4. It is therefore an ideal model in which preclinical studies can be performed. In particular this methodology may be useful to researchers interested in evaluating different devices and biologics, including stem cells, for potential application in human spinal surgery.
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Emerging evidence suggests human amnion tissue as a valuable source of two distinct types of pluripotent cells, amnion epithelial cells (hAECs) and mesenchymal stromal cells (hAMSCs), for applications in cell replacement therapy. For some approaches, it may be necessary to culture and differentiate these cells before they can be transplanted. No systematic attempt has been yet made to determine the quantity and quality of amnion cells after isolation and culture. We looked at amnion cell isolates from 27 term placentas. Following our optimized protocol, primary yields were 6.3 × 106 hAECs and 1.7 × 106 hAMSCs per gram amnion. All 27 cases gave vital cultures of hAMSCs, while one third of cases (9 of 27) failed to give adherent cultures of hAECs. Primary cultures contained significantly more proliferating than apoptotic cells (hAECs: 16.4% vs. 4.0%; hAMSCs: 9.5% vs. 2.4%). Neither hAECs nor hAMSCs were clonogenic. They showed slow proliferation that almost stopped beyond passage 5. Microscopic follow-up revealed that hAEC morphology gradually changed towards mesenchymal phenotype over several passages. Flow cytometric characterization of primary cultures showed expression of mesenchymal progenitor markers CD73, CD90, CD105, and CD166, as well as the embryonic stem cell markers SSEA-3 and -4 on both amnion cell types. These profiles were grossly maintained in secondary cultures. Reverse transcriptase-PCR analysis exhibited transcripts of Oct-3/4 and stem cell factor in primary and secondary cultures of all cases, but no telomerase reverse transcriptase. Immunocytochemistry confirmed translation into Oct-3/4 protein in part of hAEC cultures, but not in hAMSCs. Further, both amnion cell types stained for CD90 and SSEA-4. Osteogenic induction studies with amnion cells from four cases showed significantly stronger differentiation of hAECs than hAMSCs; this capacity to differentiate greatly varied between cases. In conclusion, hAECs and hAMSCs in culture exhibit and maintain a similar marker profile of mesenchymal progenitors. hAECs were found as a less reliable source than hAMSCs and altered morphology during subculture.
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Growth factors have proven to promote spine fusion. However, no comparative evaluation of growth factors in spinal fusion has yet been performed. The purpose of this study was to compare the efficacy and safety of combined IGF-I and TGF-ss1 application with BMP-2 application and autologous cancellous bone graft at an early time point in a sheep cervical spine fusion model. Thirty-two sheep underwent C3/4 discectomy and fusion. They were divided into four groups, according to their treatment: group 1, titanium cage ( n=8); group 2, titanium cage filled with autologous cancellous iliac crest bone grafts ( n=8); group 3, titanium cage coated with a poly-(D,L-lactide) (PDLLA) carrier including BMP-2 (5% w/w) ( n=8); group 4, titanium cage coated with a PDLLA carrier including IGF-I (5% w/w) and TGF-ss1 (1% w/w) ( n=8). Blood samples, body weight and temperature were analysed. Radiographic scans were performed pre- and postoperatively and after 1, 2, 4, 8 and 12 weeks. At the same time points, disc space height and intervertebral angle were measured. After 12 weeks, the animals were killed and fusion sites were evaluated using functional radiographic views in flexion and extension. Quantitative computed tomographic scans were performed to assess bone mineral density, bone mineral content and bony callus volume. Biomechanical testing was carried out and the values for range of motion, and neutral and elastic zone were determined. Histomorphological and histomorphometrical analysis were performed and polychrome sequential labelling was used to determine the time frame of new bone formation. The results showed that, in comparison to the group treated with the cage alone (group 1), the cage plus BMP-2 group (group 3) and the cage plus IGF-I and TGF-ss1 group (group 4) demonstrated a significantly higher fusion rate in radiographic findings, a higher biomechanical stability, a more advanced interbody fusion in histomorphometrical analysis, and an accelerated interbody fusion on fluorochrome sequence labelling. In comparison to the bone graft group (group 2), the BMP-2 (group 3) and IGF-I/TGF-ss1 group (group 4) showed significantly less residual motion on functional radiographic evaluation, higher bone mineral density of the callus and higher biomechanical stability in extension, rotation and bending. The BMP-2 group showed significantly less residual motion on functional radiographic evaluation and higher intervertebral bone matrix formation on fluorochrome sequence labelling at 9 weeks in comparison to the IGF-I/TGF-ss1 group. In contrast, the IGF-I/TGF-ss1 group showed a significantly higher bone mineral density of the callus than the BMP-2 group. In comparison to the autologous cancellous bone graft group, both growth factors (BMP-2 and combined IGF-I and TGF-ss1) significantly improved the biomechanical results of interbody fusion. No systemic side effects were observed for either growth factor. On the basis of these preliminary results, it would appear that combined IGF-I/TGF-ss1 application yields equivalent results to BMP-2 application at an early time point in anterior sheep cervical spine fusion.
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Previous studies have provided evidence for the existence of adult human bone marrow stromal stem cells (BMSSCs) or mesenchymal stem cells. Using a combination of cell separation techniques, we have isolated an almost homogeneous population of BMSSCs from adult human bone marrow. Lacking phenotypic characteristics of leukocytes and mature stromal elements, BMSSCs are non-cycling and constitutively express telomerase activity in vivo. This mesenchymal stem cell population demonstrates extensive proliferation and retains the capacity for differentiation into bone, cartilage and adipose tissue in vitro. In addition, clonal analysis demonstrated that individual BMSSC colonies exhibit a differential capacity to form new bone in vivo. These data are consistent with the existence of a second population of bone marrow stem cells in addition to those for the hematopoietic system. Our novel selection protocol provides a means to generate purified populations of BMSSCs for use in a range of different tissue engineering and gene therapy strategies.
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Adult bone marrow derived mesenchymal stem cells offer the potential to open a new frontier in medicine. Regenerative medicine aims to replace effete cells in a broad range of conditions associated with damaged cartilage, bone, muscle, tendon and ligament. However the normal process of immune rejection of mismatched allogeneic tissue would appear to prevent the realisation of such ambitions. In fact mesenchymal stem cells avoid allogeneic rejection in humans and in animal models. These finding are supported by in vitro co-culture studies. Three broad mechanisms contribute to this effect. Firstly, mesenchymal stem cells are hypoimmunogenic, often lacking MHC-II and costimulatory molecule expression. Secondly, these stem cells prevent T cell responses indirectly through modulation of dendritic cells and directly by disrupting NK as well as CD8+ and CD4+ T cell function. Thirdly, mesenchymal stem cells induce a suppressive local microenvironment through the production of prostaglandins and interleukin-10 as well as by the expression of indoleamine 2,3,-dioxygenase, which depletes the local milieu of tryptophan. Comparison is made to maternal tolerance of the fetal allograft, and contrasted with the immune evasion mechanisms of tumor cells. Mesenchymal stem cells are a highly regulated self-renewing population of cells with potent mechanisms to avoid allogeneic rejection.
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Stem cells capable of differentiating to multiple lineages may be valuable for therapy. We report the isolation of human and rodent amniotic fluid-derived stem (AFS) cells that express embryonic and adult stem cell markers. Undifferentiated AFS cells expand extensively without feeders, double in 36 h and are not tumorigenic. Lines maintained for over 250 population doublings retained long telomeres and a normal karyotype. AFS cells are broadly multipotent. Clonal human lines verified by retroviral marking were induced to differentiate into cell types representing each embryonic germ layer, including cells of adipogenic, osteogenic, myogenic, endothelial, neuronal and hepatic lineages. Examples of differentiated cells derived from human AFS cells and displaying specialized functions include neuronal lineage cells secreting the neurotransmitter L-glutamate or expressing G-protein-gated inwardly rectifying potassium channels, hepatic lineage cells producing urea, and osteogenic lineage cells forming tissue-engineered bone.
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Multipotent mesenchymal stromal cells isolated from bone marrow and other sites are currently being studied to determine their potential role in the pathogenesis and/or management of autoimmune diseases. In vitro studies have shown that they exhibit a dose-dependent antiproliferative effect on T and B lymphocytes, dendritic cells, natural killer cells and various B cell tumour lines--an effect that is both cell contact and soluble factor dependent. Animal models of autoimmune disease treated with multipotent mesenchymal stromal cells have mostly exhibited a positive clinical response, as have a limited number of patients suffering from acute graft versus host disease. This review summarizes the findings of a 1-day meeting devoted to the subject with the aim of coordinating efforts.
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The amnion is the inner of two membranes surrounding the fetus. That it arises from embryonic epiblast cells prior to gastrulation suggests that it may retain a reservoir of stem cells throughout pregnancy. We found that human amniotic epithelial cells (hAECs) harvested from term-delivered fetal membranes express mRNA and proteins present in human embryonic stem cells (hESCs), including POU domain, class 5, transcription factor 1; Nanog homeobox; SRY-box 2; and stage-specific embryonic antigen-4. In keeping with possible stem cell-like activity, hAECs were also clonogenic, and primary hAEC cultures could be induced to differentiate into cardiomyocytic, myocytic, osteocytic, adipocytic (mesodermal), pancreatic, hepatic (endodermal), neural, and astrocytic (neuroectodermal) cells in vitro, as defined by phenotypic, mRNA expression, immunocytochemical, and/or ultrastructural characteristics. However, unlike hESCs, hAECs did not form teratomas upon transplantation into severe combined immunodeficiency mice testes. Last, using flow cytometry we have shown that only a very small proportion of primary hAECs contain class IA and class II human leukocyte antigens (HLAs), consistent with a low risk of tissue rejection. However, following differentiation into hepatic and pancreatic lineages, significant proportions of cells contained class IA, but not class II, HLAs. These observations suggest that the term amnion, an abundant and easily accessible tissue, may be a useful source of multipotent stem cells that possess a degree of immune privilege.
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Tissue engineering techniques have been proven effective in bone regeneration and repairing load-bearing bone defects. Previous studies, however, have heretofore been limited to the use of slowdegradable or natural biomaterials as scaffolds. There are, however, no reports on using biodegradable, synthetic beta-tricalcium phosphate (β-TCP) as scaffolds to repair weight-bearing bone defects in large animals. In the present study, highly porous β-TCP scaffolds prepared by the polymeric sponge method were used to repair goat tibial defects. Fifteen goats were randomly assigned to one of three groups, and a 26 mm-long defect at the middle part of the right tibia in each goat was created. In Group A (six goats), a porous β-TCP ceramic cylinder that had been loaded with osteogenically induced autologous bone marrow stromal cells (BMSCs) was implanted in the defect of each animal. In Group B (six goats), the same β-TCP ceramic cylinder without any cells loaded was placed in the defect. In Group C (three goats), the defect was left untreated. In Group A, bony union can be observed by gross view, X-ray and micro-computed tomography (Micro-CT) detection, and histological observation at 32 weeks post-implantation. The implanted β-TCP scaffolds were almost completely replaced by tissue-engineered bone. Bone mineral density in the repaired area of Group A was significantly higher (p 0.05). In Group C, little or no new bone was formed, and non-union occurred, showing that the 26 mm segmental defect of the goat tibia was critical sized at 32 weeks. Thus, it can be concluded that the mechanical properties of the BMSCs/β-TCP composites could be much improved via tissue engineering approach and β-TCP might be used to repair the weight-bearing segmental defects of goat tibias.
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Study Design. A sheep cervical spine interbody fusion model was used to determine the effect of combined insulin-like growth factor-I (IGF-I) and transforming growth factor-beta-1 (TGF-β1) applied by a poly-(D,L-lactide) (PDLLA)-coated cage. Objectives. The purpose of this study was to determine the effect of a new PDLLA carrier system, and to evaluate the effect of combined IGF-I and TGF-β1 application in a sheep cervical spine model. Summary and Background Data. Growth factors such as bone morphogenic protein-2 have been shown to promote spine fusion and to overcome the disadvantages of an autologous bone graft. The optimum growth factor for promoting spinal fusion and the optimum method for delivering such growth factors are still a matter of discussion. Method. In this study, 32 sheep underwent C3–C4 discectomy and fusion: Group 1 (autologous tricortical iliac crest bone graft; n = 8), Group 2 (titanium cage; n = 8), Group 3 (titanium cage coated with a PDLLA carrier; n = 8), and Group 4 (titanium cage coated with a PDLLA carrier including IGF-I [5% w/w] and TGF-β1 [1% w/w; n = 8). Blood samples, body weight, and body temperature were analyzed. Radiographic scans were performed before and after surgery, then at 1, 2, 4, 8, and 12 weeks, respectively. At the same time points, the disc space height, intervertebral angle, and lordosis angle were measured. After 12 weeks, the animals were killed, and fusion sites were evaluated using functional radiographic views of the animals in flexion and extension. Quantitative computed tomographic scans were performed to assess bone mineral density, bone mineral content, and bony callus volume. Biomechanical testing of the motion segment C3–C4 was performed in flexion, extension, axial rotation, and lateral bending. The stiffness, range of motion, neutral zone, and elastic zone were determined. Histomorphologic and histomorphometric analysis was performed, and polychrome sequential labeling was used to determine the time frame of new bone formation. Results. There were no differences between the groups in terms of blood counts, body weight, and temperature. Over a 12-week period, cage Groups 2 to 4 showed significantly higher values for the intervertebral angle than for the bone graft. Functional radiographic assessment showed significantly lower residual flexion–extension movement in Group 4 than in any other group. The PDLLA-coated cages with IGF-I and TGF-β1 showed significantly higher values for bone mineral density, bone mineral content, and bony callus volume. The average stiffness in rotation and bending was significantly higher, and the range of motion, neutral zone, and elastic zone in rotation were significantly lower in Group 4 than in any other group. Although only one animal in Group 4 demonstrated solid bony fusion after 12 weeks, histomorphometric evaluation showed a more progressed bone matrix formation in the group that had PDLLA-coated cages with IGF-I and TGF-β1 than in any other group. Polychrome sequential labeling showed accelerated intervertebral bone matrix formation in Group 4. Conclusions. The findings showed that PDLLA coating of cervical spine interbody fusion cages as a delivery system for growth factors was effective. Although IGF-I and TGF-β1 application by a PDLLA-coated interbody cage was not able to achieve solid bony fusion during the 12-week follow-up period, these growth factors significantly increased the results of interbody bone matrix formation. Additional longer-term studies are required to determine whether combined IGF-I and TGF-β1 application leads to a successful spinal fusion.
Article
Bone morphogenetic proteins (BMPs) are increasingly used in spinal fusion surgery. Previous reports of BMP use in anterior cervical fusion have suggested high rates of complications related to soft tissue swelling. We evaluate the safety of using BMP-7 osteogenic protein (OP-1®; Stryker, Kalamazoo, MI, USA) in a relatively contained form and controlled dose. A prospective consecutive cohort of 131 patients underwent anterior cervical discectomy and fusion using interbody cages. In 123 of these patients, BMP-7 was also used. The primary outcome measure was the presence (or otherwise) of clinical adverse events during the first 30 days. The secondary outcome was the extent of radiological soft tissue swelling as measured on plain radiographs in the early post-operative period. There was no mortality and no reoperation in this series; however, 2.4% of patients experienced complications of transient brachalgia (1 patient), and dysphagia (2 patients). The use of BMPs in spinal fusion is discussed, and the relevant literature reviewed, particularly as it relates to adverse clinical events. We concluded that BMP-7 can be used safely in anterior cervical fusion. The effect of BMP-7 on the rate and timing of fusion, as well as clinical outcome, is yet to be elucidated.
Article
An experimental study using a sheep cervical spine interbody fusion model. To compare allogeneic mesenchymal precursor cells combined with hydroxyapatite and tricalcium phosphate (HA/TCP) with HA/TCP alone or iliac crest autograft (AG) for cervical interbody fusion. We investigated the effect of mesenchymal precursor cells on cervical fusion because of the shortcomings of using iliac crest (donor site morbidity), bone substitute (poor osteoinductive properties), and bone morphogenic proteins (serious complications). Thirty ewes were divided randomly into four groups of six having C3-C4 anterior cervical discectomy and fusion using a Fidji cage packed with, AG, HA/TCP, HA/TCP containing 5 million MPCs, and HA/TCP containing 10 million MPCs. MPCs were derived from a single batch of immuno-selected and culture-expanded MPCs isolated from bone marrow of out-bred sheep. The fifth group were nonoperated controls. Safety, fusion parameters, and biomechanics were assessed. No cell-related adverse events were observed. No significant differences were found between the five or 10 million MPC groups. Evaluation of fusion by CT scan at 3 months showed that 9 of 12 (75%) MPC-treated animals had continuous bony bridging compared with only 1 of 6 AG and 2 of 6 HA/TCP (P = 0.019 and P = 0.044, respectively). By quantitative CT, density of new bone in MPC-treated animals was 121% higher than in HA/TCP (P = 0.017) and 128% higher than in AG (P < 0.0001). Functional radiology at 3 months revealed that MPC-treated animals had significantly reduced macromotion at C3/4 compared with AG and HA/TCP groups combined (P = 0.007). Implantation of allogeneic MPCs when combined with HA/TCP and an interbody spacer facilitates new bone formation after discectomy without any cell-related complications. The earlier and dense new bone formation observed with MPCs relative to autograft and HA/TCP alone suggest that this approach may offer therapeutic benefit.
Article
The objective of this systematic review was to use evidence-based medicine to determine the efficacy of interbody graft techniques. The National Library of Medicine and Cochrane Database were queried using MeSH headings and keywords relevant to cervical interbody grafting. Abstracts were reviewed and studies that met the inclusion criteria were selected. The guidelines group assembled an evidentiary table summarizing the quality of evidence (Classes I-III). Disagreements regarding the level of evidence were resolved through an expert consensus conference. The group formulated recommendations that contained the degree of strength based on the Scottish Intercollegiate Guidelines network. Validation was done through peer review by the Joint Guidelines Committee of the American Association of Neurological Surgeons/Congress of Neurological Surgeons. Autograft bone harvested from the iliac crest, allograft bone from either cadaveric iliac crest or fibula, or titanium cages and rectangular fusion devices, with or without the use of autologous graft or substitute, have been successful in creating arthrodesis after 1- or 2-level anterior cervical discectomy with fusion (Class II). Alternatives to autograft, allograft, or titanium cages include polyetheretherketone cages and carbon fiber cages (Class III). Polyetheretherketone cages have been used successfully with or without hydroxyapatite for anterior cervical discectomy with fusion. Importantly, recombinant human bone morphogenic protein-2 carries a complication rate of up to 23-27% (especially local edema) compared with 3% for a standard approach. Current evidence does not support the routine use of interbody grafting for cervical arthrodesis. Multiple strategies for interbody grafting have been successful with Class II evidence supporting the use of autograft, allograft, and titanium cages.
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Discovered more than 40 years ago, the biological features of multipotent mesenchymal stromal cells (MSC) were progressively compared first with hematopoietic stem cells (HSC) and, more recently, with embryonic stem cells (ESC). Although these comparisons have been crucial in helping to clarify their nature, there is now a robust amount of data indicating that MSC in vitro represent an independent and heterogeneous group of progenitors with distinct self-renewal properties and established differentiation potentials. However, research developments both in humans and animals have progressively revealed the limits that MSC may face in vivo. To recognize these issues and challenge MSC stemness may seem to be a step backward. Nevertheless, it might also represent the beginning of a phase in which the introduction of novel preclinical approaches could provide better characterization and standardization of the in vivo factors influencing cell engraftment and survival, allowing a more successful impact of mesenchymal progenitors in several clinical settings.
Article
Since discovery, significant interest has been generated in the potential application of mesenchymal stem cells or multipotential stromal cells (MSC) for tissue regeneration and repair, due to their proliferative and multipotential capabilities. Although the sheep is often used as a large animal model for translating potential therapies for musculoskeletal injury and repair, the characteristics of MSC from ovine bone marrow have been inadequately described. Histological and gene expression studies have previously shown that ovine MSC share similar properties with human and rodents MSC, including their capacity for clonogenic growth and multiple stromal lineage differentiation. In the present study, ovine bone marrow derived MSCs positively express cell surface markers associated with MSC such as CD29, CD44 and CD166, and lacked expression of CD14, CD31 and CD45. Under serum-deprived conditions, proliferation of MSC occurred in response to EGF, PDGF, FGF-2, IGF-1 and most significantly TGF-alpha. While subcutaneous transplantation of ovine MSC in association with a ceramic HA/TCP carrier into immunocomprimised mice resulted in ectopic osteogenesis, adipogenesis and haematopoietic-support activity, transplantation of these cells within a gelatin sponge displayed partial chondrogenesis. The comprehensive characterisation of ovine MSC described herein provides important information for future translational studies involving ovine MSC.
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The ability of stem cells to differentiate into multiple cell lineages has ushered in exciting possibilities for stem cell based therapies that would be used to regenerate and repair damaged tissues and organs. Stem cells isolated from the embryo, fetus, adult and also the umbilical cord and placenta are being widely tested. Recent studies show that human fetal membranes also harbour cells with stem cell like properties. The amnion and chorion contain stromal cells that display characteristics and differentiation potential similar to that of adult, bone marrow derived mesenchymal stem cells. Amniotic epithelial cells share some of the features of pluripotent embryonic stem cells and multipotent mesenchymal stem cells and differentiate into multiple cell lineages in vitro. Amniotic epithelial cells also produce numerous substances that could augment tissue regeneration and repair. This review will focus on the stem cell like properties of stromal and epithelial cells derived from human fetal membranes and their potential use in stem cell based therapies.
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Four decades after the first isolation and characterization of clonogenic bone marrow stromal cells or mesenchymal stem cells (MSC) in the laboratory of Dr. Alexander Friedenstien, the therapeutic application of their progeny following ex vivo expansion are only now starting to be realized in the clinic. The multipotency, paracrine effects, and immune-modulatory properties of MSC present them as an ideal stem cell candidate for tissue engineering and regenerative medicine. In recent years it has come to light that MSC encompass plasticity that extends beyond the conventional bone, adipose, cartilage, and other skeletal structures, and has expanded to the differentiation of liver, kidney, muscle, skin, neural, and cardiac cell lineages. This review will specifically focus on the skeletal regenerative capacity of bone marrow derived MSC alone or in combination with growth factors, biocompatible scaffolds, and following genetic modification.
Article
Background context: The use of nonsteroidal anti-inflammatory drugs (NSAIDs) had been implicated as a risk factor for nonunion in spinal fusion. Even with a number of animal and human studies suggesting NSAIDs either delay or inhibit spinal fusion rates, these drugs continue to be used because of demonstrated benefits. Diclofenac sodium is no exception. It is very popular as a first-line treatment for acute pain, even perioperatively for spine fusion patients. Review of published literature reveals no studies done on the effect of diclofenac sodium on spine fusion rates. Purpose: To determine if diclofenac sodium is a risk factor for delayed union and nonunion in adult patients who underwent one- to two-level instrumented posterior lumbar interbody fusion (PLIF) using only local autogenous bone graft. Study design/setting: Retrospective, analytical. Patient sample: Two hundred and seventy-three adult patients who underwent one- to two-level PLIF with minimum 2-year follow-up. Outcome measures: Diclofenac sodium intake, fusion status. Methods: Medical records and radiographs of 273 adult patients who underwent one- to two-level PLIF in a single institution from 1999 to 2004 were reviewed. All patients did not use any NSAID or steroid perioperatively, except for diclofenac sodium postoperatively. The amount of diclofenac sodium used was quantified for the first 14 days post-op. These patients were followed-up periodically for 2 years. Demographic data, levels fused, and NSAID intake were correlated to union status. Results: Nineteen out of the 273 patients had no diclofenac sodium intake (no-dose group), 168 patients used less than or equal to 300 mg (moderate-dose group), and 86 patients used more than 300 mg of diclofenac sodium (high-dose group). There were 4 nonunions and 41 delayed unions seen. No significant difference was seen when fusion status was compared with age, sex, L5-S1 versus other levels, and smoking history (all comparisons, p>.05). There was a significant difference between one- and two-level fusions in terms of union status (p=.002). There were no nonunions and 18 delayed unions seen in 217 patients in the single-level fusion group. All 4 nonunions and 23 delayed unions were noted among the 56 patients in the two-level group. There were no cases of delayed union and nonunion in the no-dose group. There were also no nonunions but 16 delayed unions in the moderate-dose group. There were 4 nonunions and 25 delayed unions in the high-dose group. As diclofenac sodium intake increases, the incidence of delayed union and nonunion were also seen to increase (p<.001). Time to union was correlated to the amount of diclofenac sodium intake using Pearson's correlation coefficient (r=0.271, p<.001). When the patients were divided into one- and two-level fusion groups, the amount of diclofenac sodium used still had a significant positive correlation to frequency of delayed union and nonunion and in time to union in each group (all comparisons, p<.05). Conclusions: Diclofenac sodium showed a dose-dependent inhibitory effect toward spine fusion especially when used during the immediate postoperative period. No significant correlation was seen between age, sex, L5-S1 versus other levels fused and smoking history when compared with spine nonunion. Two-level lumbar fusions also showed a significant negative correlation to spine fusion compared with single-level fusions.
Article
Four-point bending was used to apply pure extension and flexion moments to the ligamentous lumbosacral spine and pelvic girdle of monkey (Macaca fascicularis), rabbit (domestic and wild, Oryctolagus cuniculus), badger (Meles meles), wallaby (Wallabia rufogrisea frutica), sheep (Ovis aries), seal (Phoca vitulina) and tiger (Panthera tigris). The absolute ranges of angular change in lumbar-lumbar joints (from X-radiographs) were considerable and similar in monkey and wallaby (greater in flexion) and in rabbit and badger (symmetrical in extension and flexion). Mass-specific bending comparisons showed that monkey and seal joints were the most and least resistant, respectively, to these moments. The patterns of mobility showed no clear scaling effects. Subsequently, additional ligamentous joint complexes (three vertebrae and two intervertebral discs) of monkey, wallaby, tiger, jaguar (Panthera onca) and seal (Halichoerus grypus) were subjected to cyclic extension and flexion moments. Changes in intervertebral angle (y, from X-radiographs) were modelled as functions of applied specific bending moments (x):y=A(1-e-Bx). A and B values represented bending capacities and joint compliances respectively. Homologous monkey and wallaby joints had considerable flexion capacities, with low compliances. Homologous jaguar and tiger joints had limited flexion capacities, but greater compliances. The data suggest that flexion resistance may be controlled by different mechanisms in different species.
Article
The Alpine goat model for multilevel anterior cervical discectomy and fusion was used to analyze the use of an intervertebral fusion device to promote an arthrodesis after anterior cervical discectomy. Comparisons were drawn with biomechanical, histologic, and radiographic data. To analyze the use of an intervertebral fusion device, with and without a bone graft substitute, to promote an arthrodesis anterior cervical discectomy. In previous studies, the goat cervical spine has proven to be an excellent model for examining the healing of fusions using bone grafts, instrumentation, or bone substitutes. Three-level anterior cervical dissectomies were performed on 21 mature Alpine goats. Three treatment groups of seven goats each were used. Group I used a standard titanium cervical BAK device filled with autogenous bone graft. Group II used a hydroxyapatite-coated BAK device filled with autogenous bone graft. Group III used a BAK device filled with recombinant human bone morphogenetic protein-2. Radiographically, no cages became displaced. Lucencies were seen around 3 of the 21 cages in Group 1, 4 cages in Group II, and none in Group III. Fluorochrome analysis revealed that the recombinant human bone morphogenetic protein-2-filled cages had an accelerated rate of bone growth around and through each cage-vertebral body interface at 3 weeks. A successful arthrodesis was also more likely with a recombinant human bone morphogenetic protein-2-filled cage (95%) than the hydroxyapatite-coated (62%) or the standard (48%) cage. Biomechanical stiffness testing did not reveal any statistically significant differences between the three groups. There was a tendency for successfully arthrodesed interspaces to be stiffer than those that were not. The use of a threaded intervertebral fusion cage, with or without hydroxyapatite coating, filled with autogenous bone graft provides a fusion rate that is slightly better than those previously reported using autogenous interbody bone grafts with or without plate stabilization. Recombinant human bone morphogenetic protein-2-filled cages resulted in a much higher arthrodesis rate and accelerated bone formation compared with either autogenous bone-filled BAK devices, or autogenous interbody bone grafts with or without plate stabilization.
Article
This is a follow-up study of a multicenter, randomized, placebo-controlled clinical trial conducted in accordance with the condition for Food and Drug Administration approval for pulsed electromagnetic fields. The purpose of this study was to evaluate the long-term efficacy and safety of pulsed electromagnetic fields for spinal fusion. An earlier clinical trial study was conducted to evaluate the efficacy of Pulsed Electromagnetic Fields to enhance fusion success at one year follow-up. In the original study, 195 patients undergoing interbody fusion were enrolled. Of the 195 patients, 98 were in the active group and 97 were in the placebo group. Study results showed a 92% successful fusion rate in the active group compared to 68% in the placebo group. For this long-term follow-up study, all patients who had healed in the original study were recalled for a follow-up radiograph. Radiographs were assessed by the attending surgeon for fusion assessment, when possible. The results of this long-term follow-up study showed that there was a reduction in maintenance of the fusion over time by 25%, but that the reduction was unrelated to treatment group and correlated statistically with whether the patient was a smoker.
Article
We have recently found that human amniotic epithelial (HAE) cells synthesize catecholamines including dopamine (DA). The present study was designed to explore the possibility of HAE cells to serve as a donor for transplantation therapy of Parkinson's disease (PD). Thus, we investigated their ability to produce DA in vitro and the survival and function of HAE cells grafted into a rat model of PD. RT-PCR and Western blotting revealed that HAE cells express tyrosine hydroxylase (TH) mRNA and protein, respectively. TH-immunohistochemistry on cultured HAE cells demonstrated that around 10% of the total cells are immunopositive for this protein. The production of DA by HAE cells was increased with time in the presence of L-tyrosine and BH(4), and was abolished with a specific TH inhibitor, alpha-methyl-rho-tyrosine. Dissociated HAE cells transduced with the Escherichia coli LacZ marker gene (beta-gal) were implanted into the previously DA-depleted striatum of immunosuppressed rats. Two weeks postgrafting HAE grafts were demonstrated to survive without overgrowth, as evidenced by the presence of beta-gal-positive cells and TH-immunoreactive cells within the grafts. The grafts also provided partial amelioration of apomorphine-induced rotational asymmetry. The results clearly indicate that HAE cells capable of producing DA can survive and function in the brain of a rat model of PD. Although DA replacement therapy of PD could possibly be achieved with implantation of HAE cells, further studies are needed to develop strategies to enhance the ability of HAE cells to produce DA as well as the graft survival.
Article
A review of the smoking habits in 426 patients who had been followed prospectively for 2 years after a lumbar spinal fusion procedure was conducted. To analyze the effect of pre- and postoperative smoking on clinical and functional outcome after lumbar spinal fusion. Several animal models have shown a negative effect of nicotine on spinal fusion. At this writing, the clinical effect of nicotine on spinal fusion has not been fully clarified. The study comprised 426 patients who underwent lumbar spinal fusion between 1993 and 1997. These patients received a mailed questionnaire regarding their tobacco consumption before and after their surgery. All other data, including preoperative clinical and functional status, were collected prospectively during a 2-year follow-up period. To assess functional outcome, the Dallas Pain Questionnaire was used. The questionnaire was answered by 396 patients (93%). Of these patients, 54.5% (20% more than the background population) were smokers before the operation. Smoking of more than 10 cigarettes daily before the operation and attempted fusion at two or more levels increased the risk of nonunion: odds ratio, 2.01 (P < 0.016) and odds ratio, 3.03 (P < 0.001), respectively. Smoking cessation increased fusion rates to near those of nonsmokers. Smoking had no influence on functional outcome, as assessed by the Dallas Pain Questionnaire, but preoperative smoking predicted a negative answer to the question "Would you undergo the same treatment again, now that you know the result?" (odds ratio, 1.65; P < 0.054). Smoking was shown to have a negative effect on fusion and overall patient satisfaction, but no measurable influence on the functional outcome as assessed by the Dallas Pain Questionnaire.
Article
A prospective randomized study investigated the radiographic progress of fusion at 6, 12, and 24 months in 42 patients who underwent a single-level anterior lumbar interbody fusion using cylindrical interbody fusion cages. To determine the patterns and rates of osteoinduction associated with the use of recombinant human bone morphogenetic protein type 2 (rhBMP-2) and an absorbable collagen sponge carrier in anterior lumbar interbody fusion with a tapered cylindrical fusion device. Studies have shown that rhBMP-2 used with allograft dowels increases the rate of interbody fusion by promoting osteoinduction and enhancing incorporation of the allograft. In a small series of human patients undergoing anterior lumbar interbody fusion with a tapered cylindrical fusion cage, rhBMP-2 has been shown to promote osteoinduction and fusion. In this prospective nonblinded study, 42 patients were randomly divided into two groups. The investigational group underwent interbody fusion using two tapered cylindrical fusion cages (LT-CAGE) and rhBMP-2 on an absorbable collagen sponge, and a control group underwent the procedure, receiving the devices and autogenous iliac crest bone graft. Plain radiographs and computed tomographic scans were used to evaluate the pattern of osteoinduction in the interbody space and the progression of fusion 6, 12, and 24 months after surgery. All the patients who received rhBMP-2 showed radiographic evidence of osteoinduction in the interbody cages 6 months after surgery. The density in the cages had increased an average of 142 Hounsfield units by 6 months. At 12 months, the increase had reached 228.7 Hounsfield units New bone formation occurred in the disc space outside the cages by 6 months in 18 of the patients in the investigational group (18/22; 82%). By 24 months, all the investigational patients showed new formation outside the cages. In the autograft control group, the density in the cages increased an average of 42 Hounsfield units, and 10 patients (10/20; 50%) showed evidence of bone formation outside the cages. The use of rhBMP-2 is a promising method for facilitating anterior intervertebral spinal fusion in patients who have undergone anterior lumbar fusion surgery.
Article
A prospective, randomized, pilot clinical trial compared recombinant human bone morphogenetic protein-2 (rhBMP-2) with iliac crest autograft bone for the treatment of human cervical disc disease. To examine the safety and effectiveness of using INFUSE Bone Graft (rhBMP-2 applied to an absorbable collagen sponge), as compared with an autogenous iliac crest bone graft placed inside the CORNERSTONE-SR fibular allograft, in anterior cervical discectomy and interbody fusion. Recombinant human bone morphogenetic protein-2 is an osteoinductive protein that induces a reliable fusion in the lumbar spine, but it has not been studied in patients with degenerative cervical disc disease. For this study, 33 patients with degenerative cervical disc disease were randomly assigned to investigational or control groups. The investigational group received a fibular allograft (CORNERSTONE-SR Allograft Ring) with an rhBMP-2-laden collagen carrier inside the graft along with an ATLANTIS anterior cervical plate. The control group received a fibular allograft with cancellous iliac crest autograft placed inside it, along with an ATLANTIS anterior cervical plate. The patients underwent plain radiographs at 6 weeks, then at 3, 6, 12, and 24 months, and CT scans at 3 and 6 months after surgery. They also completed general health profiles and self-evaluation scales. Adverse events were evaluated for severity, duration, association with the implant, and the need for a second surgical procedure. All the patients evaluated had solid fusions 6, 12, and 24 months after surgery. There were no device-related adverse events. At 24 months, the investigational group had mean improvement superior to that of the control group in neck disability and arm pain scores (P < 0.03 each). This pilot study demonstrates the feasibility of using rhBMP-2 safely and effectively in the cervical spine.
Article
We recently found that human amniotic epithelial (HAE) cells secrete biologically active neurotrophins such as brain-derived neurotrophic factor and neurotrophin-3, both of which exhibit trophic activities on dopamine (DA) neurons. The present study explored whether implantation of HAE cells can be a possible means to deliver trophic factors into the brain to prevent the death of DA neurons in a rat model of Parkinson's disease. We first investigated the ability of HAE cells to produce factors capable of promoting DA cell survival in vitro, and then tested whether HAE cell grafts survive and prevent the death of nigral DA neurons in rats with 6-hydroxydopamine lesions. A treatment with conditioned medium derived from HAE cell cultures enhanced the survival of tyrosine hydroxylase (TH)-immunopositive DA cells in serum-free cultures. The conditioned medium also protected the morphological integrity of TH-positive neurons against toxic insult with 6-hydroxydopamine. HAE cells were grafted into the midbrain of immunosuppressed rats. The rats were then subjected to a unilateral nigrostriatal lesion induced by intrastriatal infusions of 6-hydroxydopamine. HAE cell transplants were found to survive without evidence for overgrowth 2 weeks postgrafting. The number of nigral DA cells, detected with either TH-immunohistochemistry or retrograde labelling with fluorogold, was significantly increased in rats given the grafts as compared to that in control animals without the grafts. The results indicate that HAE cells produce diffusible molecules that can enhance the survival of DA neurons. Although the factors that contribute to the currently observed effects remain to be fully determined, implantation of HAE cells could be a viable strategy to counteract the loss of DA neurons in Parkinson's disease.
Article
Whole pancreas or beta-cell transplantation has opened the way for the treatment of advanced stage of diabetes mellitus. However, it is always limited by the scarcity of transplantation materials. The amniotic membrane is part of the fetal membrane and is composed of amniotic epithelium (HAE) and mesenchymal (HAM) cells that are derived from the inner cell mass in the blastocyst. Thus, HAE and HAM cells may have the potential to differentiate into various organs. The aim of our study was to assess the possibility of HAE cells differentiating into insulin-producing cells. In vitro, HAE cells stimulated with nicotinamide induced insulin mRNA in the culture cells. In vivo, HAE cells were capable of normalizing the blood glucose level of diabetic mice after several weeks of implantation into streptozotocin-induced diabetic mice. The distribution of human cells and human insulin secretion in mouse tissue studied by immunohistochemistry for anti-human-specific beta-2-microglobulin and anti-human-specific insulin shows the same location in mouse tissue. These studies suggest that HAE cells have the potential to differentiate into beta-cells in vivo, and hence that HAE cells have therapeutic potential for the treatment of type I diabetes mellitus.
Article
Fetal membranes are tissues of particular interest for several reasons, including their role in preventing rejection of the fetus and their early embryologic origin. which may entail progenitor potential. The immunologic reactivity and the transplantation potential of amnion and chorion cells, however, remain to be elucidated. Amnion and chorion cells were isolated from human term placenta and characterized by immunohistochemistry, flow cytometric analysis, and expression profile of relevant genes. The immunomodulatory characteristics of these cells were studied in allogeneic and xenogeneic mixed lymphocyte reactions and their engraftment potential analyzed by transplantation into neonatal swine and rats. Posttransplant chimerism was determined by polymerase chain reaction analysis with probes specific for human DNA. Phenotypic and gene expression studies indicated mesenchymal stem cell-like profiles in both amnion and chorion cells that were positive for neuronal, pulmonary, adhesion, and migration markers. In addition, cells isolated both from amnion and chorion did not induce allogeneic nor xenogeneic lymphocyte proliferation responses and were able to actively suppress lymphocyte responsiveness. Transplantation in neonatal swine and rats resulted in human microchimerism in various organs and tissues. Human amnion and chorion cells from term placenta can successfully engraft neonatal swine and rats. These results may be explained by the peculiar immunologic characteristics and mesenchymal stem cell-like phenotype of these cells. These findings suggest that amnion and chorion cells may represent an advantageous source of progenitor cells with potential applications in a variety of cell therapy and transplantation procedures.
Article
Bone is a tissue that constantly undergoes deposition, resorption of stromal matrix, and remodeling. These processes may be altered by a variety of chemical, mechanical, cellular, and pathological mechanisms. Understanding the physiology of bone healing and the mechanisms affecting this process is important not only when evaluating normal skeletal development but also when initiating fracture repair. Because the ultimate success of spinal fusions involves creation of an osseous union, we focus this review on the anatomy and physiology of bone under physiological conditions, normal bone healing and mechanisms that alter it, and available adjuvant therapies that may enhance healing potential in a clinical setting.
Article
Degenerative disorders in the spine are normal, age-related phenomena and largely asymptomatic in most cases. Conservative management of lumbar and cervical spondylosis is the mainstay of treatment, and most patients with symptomatic degenerative changes respond appropriately with nonsurgical management. Surgical intervention can be considered an appropriate and viable option when conservative measures have failed. Treatment options should always be directed toward the specific nature and location of the patient's individual pathology. Although current standards in the surgical management of lumbar and cervical degenerative disorders include discectomy, neural decompression, and instrumented spinal arthrodesis, new approaches that address this often-challenging clinical entity are on the horizon.
Article
A retrospective review of 33 consecutive patients treated with posterior fusion and selective nerve root decompression for the treatment of pseudarthrosis following anterior cervical discectomy and fusion. Use standardized outcome measures to evaluate the results of posterior fusion with selective nerve root decompression as a treatment option for symptomatic pseudarthrosis of the cervical spine. Pseudarthrosis after anterior cervical discectomy and fusion has been recognized as a cause of continued cervical pain and unsatisfactory outcomes. Debate continues as to whether a revision anterior approach or a posterior fusion procedure is the best treatment for symptomatic cervical pseudarthrosis. To our knowledge, standardized outcome measures have not been used to evaluate the results of either surgical treatment option; therefore, it is difficult to evaluate outcomes in these patients, let alone compare surgical treatment options. Data on fusion rates in these two surgical treatment groups suggest a trend of a higher fusion rate with utilization of a posterior revision procedure, but the largest study to date includes the study of only 19 patients treated with a posterior fusion. Thirty-three consecutive patients with symptomatic pseudarthrosis following anterior cervical discectomy and fusion were treated with selective nerve root decompression and posterior fusion using iliac crest or local bone graft as well as posterior wiring and/or lateral mass plating. The average follow-up period was 46 months (range, 20-86 months). Patients were assessed using physical examination, flexion-extension lateral radiographs, and standardized outcome measures including the SF-36, Arthritis Impact Measurement Scales 2, and Cervical Spine Outcomes Questionnaire. All 33 patients (100%) demonstrated a solid fusion at their most recent follow-up, and all 33 patients noted significant improvement in their preoperative symptoms. No difference in fusion status was noted between those treated with iliac crest versus patients treated with local bone graft--all had a solid fusion; 72% of the patients were satisfied with the result of their surgery. Cervical Spine Outcomes Questionnaire pain scales demonstrated 52% of patients reported mild or nopain at follow-up, whereas 20% described their pain as "discomforting" and 28% of the patients continued to report moderate to severe pain. This is the first study to our knowledge to use standardized outcome measures to assess clinical outcome in patients treated with posterior fusion for pseudarthrosis after anterior cervical discectomy and fusion. Patients and surgeons need to understand the potential for success with this revision procedure but also be aware of the relatively high rate of continued moderate to severe pain observed in this patient population even after a solid fusion is achieved. All of the patients in this study fused with a single posterior fusion procedure, further supporting the relatively higher fusion rates observed in the literature using posterior fusion as a treatment for cervical pseudarthrosis. Our results also support the ability of surgeons to use local bone graft without iliac crest in a posterior fusion for cervical pseudarthrosis and therefore avoid the morbidity associated with iliac crest bone graft harvest.
Article
Background context: In 1965, Marshall Urist discovered that the extracellular matrix of bone contains the ability to induce new bone formation. This substance was later named bone morphogenetic protein (BMP). Since that time, BMPs have been extensively studied. Molecular clones have since been characterized and expressed as complementary DNAs (cDNAs). BMPs have recently been used in a multitude of mammalian clinical studies, including many recent human studies, for the purpose of evaluating their function in bone healing and spinal arthrodesis. BMPs are currently the most effective substitute available for bone graft as a means to eliminate the morbidity of iliac crest bone graft harvest and increase the rate of successful spinal arthrodesis. Purpose: The purpose of this article is to review the history and recent advancements in the use of BMPs in spinal arthrodesis models, as well as discuss the possible future use of BMPs in this clinical setting. Study design/setting: The setting of this review article is centered on classic and recent literature of BMPs with emphasis on anterior as well as posterolateral spinal arthrodesis. Methods: The classic and recent primary literature about BMPS and their clinical use in human and nonhuman mammals for spinal fusion was reviewed. Special emphasis is placed on animal and human studies of both recombinant human bone morphogenetic protein-2 (rhBMP-2) and recombinant human osteogenic protein-1 (rhOP-1, rhBMP-7). Results: BMPs are able to promote solid fusion in the spine in both the anterior and the posterolateral environments in animal studies and early human trials. Human trials to date have also shown an acceptable safety profile with the clinical use of these proteins. Conclusions: Animal studies and early human trials of BMPs support the ability of these growth factors to enhance or replace autograft bone for spinal arthrodesis. Studies have shown this promotion of fusion in both the anterior interbody and the posterolateral environments. Future use of these factors is likely to continue to expand in clinical as well as research arenas.
Article
Our contemporary understanding of bone healing has evolved due to knowledge gleaned from a continuous interaction between basic laboratory investigations and clinical observations following procedures to augment healing of fractures, osseous defects, and unstable joints. The stages of bone healing parallel the early stages of bone development. The bone healing process is greatly influenced by a variety of systemic and local factors. A thorough understanding of the basic science of bone healing as well as the many factors that can affect it is critical to the management of a variety of musculoskeletal disorders. In particular, the evolving management of spinal disorders can greatly benefit from the advancement of our understanding of the principles of bone healing.
Article
The considerable therapeutic potential of human multipotent mesenchymal stromal cells (MSC) has generated markedly increasing interest in a wide variety of biomedical disciplines. However, investigators report studies of MSC using different methods of isolation and expansion, and different approaches to characterizing the cells. Thus it is increasingly difficult to compare and contrast study outcomes, which hinders progress in the field. To begin to address this issue, the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy proposes minimal criteria to define human MSC. First, MSC must be plastic-adherent when maintained in standard culture conditions. Second, MSC must express CD105, CD73 and CD90, and lack expression of CD45, CD34, CD14 or CD11b, CD79alpha or CD19 and HLA-DR surface molecules. Third, MSC must differentiate to osteoblasts, adipocytes and chondroblasts in vitro. While these criteria will probably require modification as new knowledge unfolds, we believe this minimal set of standard criteria will foster a more uniform characterization of MSC and facilitate the exchange of data among investigators.
Article
Fourteen sheep were fused using anterior interbody implants at C2-C3 and C4-C5 and followed for 6 months. To evaluate the effect of absorbable and nonabsorbable implants on fusion rate, cage migration, and implant integrity. Despite the high clinical success rate with metallic plates and interbody grafting, complications such as dysphagia, imaging artifacts, and revision difficulties exist. Less permanent and lower profile implants could minimize these problems. Four treatments were studied: 1) carbon fiber-reinforced polymer (CFRP) cage alone, 2) CFRP cage with an absorbable tension band, 3) absorbable cage with an absorbable tension band, and 4) absorbable cage with a titanium plate. Fusion was assessed using radiographs, biomechanical testing, and micro-CT analysis. Treatments with the CFRP cage alone had the lowest fusion rate at 3 months (2/6) and 6 months (4/6). The CFRP cage with the absorbable strap treatments had 5/6 fusions at 6 months. The absorbable cage with absorbable strap also had 5/6 fusions, but two of the cages fractured. The absorbable cage with titanium plate had 5/6 fusions, but the one partial fusion was attributed to poor screw and plate placement. Using both absorbable and nonabsorbable implants, high fusion rates were achieved in the challenging sheep cervical spine model. However, the absorbable cages were not able to withstand the mechanical forces during the 6-month survival period.
Article
Numerous studies support the concept that the nonhemopoietic cells of the bone marrow (BM), are derived from a population of multipotent bone marrow stromal stem cells (BMSSCs), which reside in perivascular niches within the bone marrow. These BMSSCs are thought to give rise not only to more cells that are phenotypically and functionally identical but also differentiated, lineage-committed mesenchymal progeny, including chondrocytes, smooth muscle cells, adipocytes, and osteoblasts. Recently, we have generated a novel monoclonal antibody (mAb) (designated STRO-3) that reacts with a minor subset of STRO-1(+) cells contained within adult BM aspirates and does not react with CD34(+) hemopoietic stem cells. Our results also show that STRO-3 identifies a high proportion of BMSSCs that possess extensive proliferative and multilineage differentiative capacity. Using retroviral expression cloning, we determined that STRO-3 binds to tissue nonspecific alkaline phosphatase (TNSALP), a cell-surface glycoprotein usually associated with cells of the osteoblast lineage. Studies presented here suggest that in addition to being expressed by osteoblasts, TNSALP may also represent a marker of immature BMSSCs in vivo. Finally, these studies suggest that antibodies to TNSALP may be used as an effective single marker of enrichment of BMSSCs from various tissues.
Article
Although outcomes after cervical fusion in rheumatoid arthritis (RA) patients are widely published, outcomes of lumbar fusion in RA patients has not been reported. Nineteen patients with RA, identified using ICD-9 and CPT codes, who underwent instrumented posterolateral lumbar fusion were matched for age, gender, smoking status, date, and level of surgery to a contemporaneous non-RA group. Medical records and radiographs were reviewed by the primary author who had no role in the treatment of these patients. The average age was 64 years in the RA group and 65 years in the non-RA group. The male to female ratio was 2:17 and 1:18, respectively. There were three smokers and two diabetics in each group. An average of 1.5 levels was fused in each group. Average follow-up was 24 and 27 months, respectively. In the RA group, 15 patients were taking DMARDs with 7 of those also taking oral steroids; 4 patients were taking NSAIDs only. There were seven complications (37%) in the RA group versus four (21%) in the non-RA group; wound infections in three patients (16%) in the RA group versus one (5%) in the non-RA group; and non-union in two patients (11%) in the RA group versus three (16%) in the non-RA group. Clinical outcomes were similar between the two groups with 74% of patients achieving good to excellent results in the RA group compared to 63% in the non-RA group (p = 0.692). Surgeons and their RA patients who undergo an instrumented lumbar fusion can expect a slightly higher complication rate than patients without RA which may be related to osteopenia and immunosuppression.
Article
Prospective clinical trial. The goal of this prospective study was to assess the reliability of plain radiographs, computed tomography (CT), and magnetic resonance imaging (MRI) to detect a pseudarthrosis after an anterior cervical fusion compared with intraoperative exploration. Although anterior cervical fusions have been performed for >50 years, diagnosis of pseudarthrosis remains controversial, and even though various radiologic methods have been described to detect a pseudarthosis, no reports have compared these studies against the gold standard of intraoperative exploration to determine which is most accurate. All explorations were performed >6 months after the index procedure and studies were obtained within a month of surgery. Indications for reoperation were adjacent level disease and/or pseudarthrosis repair. All patients were thoroughly explored using a microscope, uncinate-to-uncinate exploration, and if necessary, removal of part of the corticalized graft. Radiologic studies were blindly and independently reviewed by 3 spine surgeons uninvolved in the care of the patients. Of the 14 patients included, 8 had pseudarthroses. Assessment of the agreement between intraoperative and radiographic findings revealed a mean Kappa statistic of 0.67 (range, 0.51-0.75, P < 0.05) for plain radiographs, 0.81 (range, 0.71-0.87, P < 0.05) for CT, and 0.48 (range, 0.32-0.71, P < 0.05) for MRI. When all studies were taken into consideration, the mean Kappa statistic increased to 0.85 (range, 0.71-1.00, P < 0.05). Assessment of paired interobserver reliability revealed a mean Kappa statistic of 0.46 (range, 0.31-0.55, P < 0.05) for plain radiographs, 0.82 (range, 0.73-0.87, P < 0.05) for CT, and 0.32 (range, 0.21-0.40, P < 0.05) for MRI. When all radiographic studies were taken into consideration, paired interobserver reliability had a mean Kappa statistic of 0.70 (range, 0.55-0.85, P < 0.05). Our results indicate that CT most closely agrees with intraoperative findings. We therefore recommend that CT be used in any study that relies on fusion status assessment. It is important to remember, however, that even CT is not 100% accurate.
Donor site morbidity after anterior iliac crest bone harvest for single-level anterior cervical discectomy and fusion
  • Js Silber
  • Dg Anderson
  • Daffner
  • Sd
Silber JS, Anderson DG, Daffner SD, et al. Donor site morbidity after anterior iliac crest bone harvest for single-level anterior cervical discectomy and fusion. Spine (Phila Pa 1976). 2003;28(2):134-139.
FDA public health notification: life-threatening complications associated with recombinant human bone morphogenetic protein in cervical spine fusion. US Food and Drug Administration, Center for Devices and Radiological Health://www.fda.gov/cdrh/safety
  • Drug Food
  • Administration
Food and Drug Administration. FDA public health notification: life-threatening complications associated with recombinant human bone morphogenetic protein in cervical spine fusion. US Food and Drug Administration, Center for Devices and Radiological Health. Accessed July 2008. http://www.fda.gov/cdrh/safety/ 070108-rhbmp.html.
Study of 3 doses of NeoFuse combined with MasterGraft Granules in subjects requiring posterolateral lumbar fusion (PLF):// clinicaltrials.gov/ct2/show/NCT00549913
  • Nih
NIH. Study of 3 doses of NeoFuse combined with MasterGraft Granules in subjects requiring posterolateral lumbar fusion (PLF). Accessed 2007. http:// clinicaltrials.gov/ct2/show/NCT00549913.
Isolation of amniotic stem cell lines with potential for therapy
  • De Coppi
  • P Bartsch
  • G Jr
  • Mm Siddiqui
De Coppi P, Bartsch G Jr, Siddiqui MM, et al. Isolation of amniotic stem cell lines with potential for therapy. Nat Biotechnol. 2007;25(1):100-106.
Allogeneic mesenchymal precursor cells safely and effectively increase the rate and robustness of cervical interbody fusion. Paper presented at: Orthopedic Research Society 55th Annual Meeting
  • T Goldschlager
  • S Itescu
  • P Ghosh
Goldschlager T, Itescu S, Ghosh P, et al. Allogeneic mesenchymal precursor cells safely and effectively increase the rate and robustness of cervical interbody fusion. Paper presented at: Orthopedic Research Society 55th Annual Meeting; 2009; Las Vegas, NV.
Comparison between sheep and human cervical spines: an anatomic, radiographic, bone mineral density, and biomechanical study
  • F Kandziora
  • R Pflugmacher
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