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Effects of substrate stiffness on adipogenic and osteogenic differentiation of human mesenchymal stem cells

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... It demonstrates good biocompatibility, biodegradability, and gel-forming properties (Tan & Marra, 2010). However, as HA is rapidly metabolised by hyaluronidases in vivo, it needs to be modified (2003), Fan et al. (2015), Mauney et al. (2007), Rowley, Madlambayan, and Mooney (1999), Tan and Marra (2010), and Zhao, Li, Liu, Zhang, and Wen (2014) Synthetic (e.g., poly(L-lactide-coglycolide) and poly(ethylene) glycol) • Chemical and mechanical properties readily to support cell growth. A potential solution are covalently cross- (Shu et al., 2004). ...
... However, it was observed that this ratio had lower cell viability than a 1:3 DAT : silk fibroin hydrogel. The authors attributed this to the theory that ADSCs were more likely to differentiate to mature adipocytes in a scaffold that more accurately mimicked native adipose tissue (Zhao et al., 2014). The addition of pre-endothelial cells to a natural scaffold such as silk fibroin demonstrated potential for the creation of a vascularised engineering adipose tissue construct (Kayabolen et al., 2017) Chitosan and chondroitin sulfate are hydrogel biomaterials that have also been utilised in adipose tissue engineering. ...
Article
Current methods of breast reconstruction are associated with significant shortcomings, including capsular contracture, infection, rupture, the need for reoperation in implant‐based reconstruction, and donor site morbidity in autologous reconstruction. These limitations result in severe physical and psychological issues for breast cancer patients. Recently, research has moved into the field of adipose tissue engineering to overcome these limitations. A wide range of regenerative strategies have been devised utilising various scaffold designs and biomaterials. A scaffold capable of providing appropriate biochemical and biomechanical cues for adipogenesis is required. Hydrogels have been widely studied for their suitability for adipose tissue regeneration, and are advantageous secondary to their ability to accurately imitate the native extracellular matrix. The aim of this review is to analyse the use of hydrogel scaffolds in the field of adipose tissue engineering.
... According to the open literature, when Young's modulus of the cartilage scaffolds is less than 4 kPa, differentiation of bone marrow MSCs into fat may happen. Furthermore, some others have suggested the [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30] kPa interval as the desirable range for Young's modulus when one tries to prepare the medium leading to differentiation of MSCs into native cells of the cartilage tissue J o u r n a l P r e -p r o o f [93][94][95]. Accordingly, the 0 wt. % Magnetite sample may not be desirable in terms of mechanical performance after implanting in the target tissue [95]. ...
... Accordingly, the 0 wt. % Magnetite sample may not be desirable in terms of mechanical performance after implanting in the target tissue [95]. Consequently, it can be suggested that scaffolds containing Magnetite are more suitable in terms of mechanical performance. ...
Article
In this study, four-phase Gelatin-Polypyrrole-Akermanite-Magnetite scaffolds were fabricated and analyzed using in-vitro tests and numerical simulations. Such scaffolds contained various amounts of Magnetite bioceramics as much as 0, 5, 10, and 15 wt% of Gelatin-Polypyrrole-Akermanite biocomposite. X-ray diffraction analysis and Fourier transform infrared spectroscopy were conducted. Swelling and degradation of the scaffolds were studied by immersing them in phosphate-buffered saline, PBS, solution. Magnetite bioceramics decreased the swelling percent and degradation duration. By immersing scaffolds in simulated body fluid, the highest formation rate of Apatite was observed in the 15 wt% Magnetite samples. The mean pore size was in an acceptable range to provide suitable conditions for cell proliferation. MG-63 cells were cultured on extracts of the scaffolds for 24, 48, and 72 h and their surfaces for 24 h. Cell viabilities and cell morphologies were assessed. Afterward, micromechanical models with spherical and polyhedral voids and artificial neural networks were employed to predict Young's moduli of the scaffolds. Based on the results of finite element analyses, spherical-shaped void models made the best predictions of elastic behavior in the 0, 5 wt% Magnetite scaffolds compared to the experimental data. Results of the simulations and experimental tests for the ten wt% Magnetite samples were well matched in both micromechanical models. In the 15 wt% Magnetite sample, models with polyhedral voids could precisely predict Young's modulus of such scaffolds.
... This hypothesis has been confirmed ever since by many other studies which are summarized in Table 1. Briefly, neurogenic [43,44,45,46] and adipogenic [33,47,48,49,50] differentiation have been found to be predominant on soft matrices (from 0.1 to 5 kPa), myogenic commitment has been shown to be mostly encouraged for stiffnesses between 8 and 40 kPa [43,45,46,47,51,52], while tenogenic differentiation was favored for stiffnesses between 30 and 50 kPa. [53] In addition, Yang et al. demonstrated that a soft matrix, with a stiffness close to that of bone marrow (2 kPa), would allow MSCs to maintain their stem cell phenotype. ...
... [53] In addition, Yang et al. demonstrated that a soft matrix, with a stiffness close to that of bone marrow (2 kPa), would allow MSCs to maintain their stem cell phenotype. [54] Finally, the results obtained for chondrogenic and osteogenic differentiation were more heterogeneous, with chondrogenic differentiation reported both on soft matrices (0.5 -1.5 kPa) [55,56] and stiffer matrices (80 kPa) [46], and osteogenic differentiation mentioned for a wide range of stiffnesses going from 1.5 up to 190 kPa [33,43,44,46,48,49,50,51,53,54,55,56,57,58,59,60], although it would be predominant between 20 and 90 kPa. Such inhomogeneity in the results could be explained by the use of a broad range of techniques to evaluate hydrogels stiffness, which complicates the comparison between the different studies. ...
... To better mimic the in vivo situation, direct coculture of these two cell types will not only be tested in conventional 2D culture but also in 3D culture, as the 3D conformation is thought to support the maturation and function of the bone cells applied to the carrier [15]. However, the choice of the carrier might be crucial, as it may affect the cells' attachment to and infiltration into the carrier, as well as the cells' differentiation [16]. Regarding the carrier, a large variety of materials have been proven to be compatible for bone tissue engineering. ...
Article
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A large British study, with almost 3000 patients, identified diabetes as main risk factor for delayed and nonunion fracture healing, the treatment of which causes large costs for the health system. In the past years, much progress has been made to treat common complications in diabetics. However, there is still a lack of advanced strategies to treat diabetic bone diseases. To develop such therapeutic strategies, mechanisms leading to massive bone alterations in diabetics have to be well understood. We herein describe an in vitro model displaying bone metabolism frequently observed in diabetics. The model is based on osteoblastic SaOS-2 cells, which in direct coculture, stimulate THP-1 cells to form osteoclasts. While in conventional 2D cocultures formation of mineralized matrix is decreased under pre-/diabetic conditions, formation of mineralized matrix is increased in 3D cocultures. Furthermore, we demonstrate a matrix stability of the 3D carrier that is decreased under pre-/diabetic conditions, resembling the in vivo situation in type 2 diabetics. In summary, our results show that a 3D environment is required in this in vitro model to mimic alterations in bone metabolism characteristic for pre-/diabetes. The ability to measure both osteoblast and osteoclast function, and their effect on mineralization and stability of the 3D carrier offers the possibility to use this model also for other purposes, e.g., drug screenings.
... Generally, substrate stiffness may likely guide MSC differentiation down corresponding tissue lineages of similar stiffness. For example, substrates approximating the elastic moduli of cartilage (0.4-0.8 MPa) may be more likely to enhance stem cells toward chondrogenesis [179][180][181], while scaffolds closely mimicking that of adipose tissue (2-8 kPa) might promote adipogenesis [176,[182][183][184]. ...
Article
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Adult stem cells, also termed as somatic stem cells, are undifferentiated cells, detected among differentiated cells in a tissue or an organ. Adult stem cells can differentiate toward lineage specific cell types of the tissue or organ in which they reside. They also have the ability to differentiate into mature cells of mesenchymal tissues, such as cartilage, fat and bone. Despite the fact that the balance has been comprehensively scrutinized between adipogenesis and osteogenesis and between chondrogenesis and osteogenesis, few reviews discuss the relationship between chondrogenesis and adipogenesis. In this review, the developmental and transcriptional crosstalk of chondrogenic and adipogenic lineages are briefly explored, followed by elucidation of signaling pathways and external factors guiding lineage determination between chondrogenic and adipogenic differentiation. An in-depth understanding of overlap and discrepancy between these two mesenchymal tissues in lineage differentiation would benefit regeneration of high-quality cartilage tissues and adipose tissues for clinical applications.
... But high ALP activity in stem cells is not only attributed to the tissue non-specific ALP, but also to other members of the ALP family [63]. In addition, there is continuous discussion, that low carrier stiffness might even favor MSC differentiation toward adipocytes [26]. Furthermore, the calcium-phosphate crystallization method additionally increased the surface roughness of the scaffold. ...
Article
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Human adipose-derived mesenchymal stem/stromal cells (Ad-MSCs) have great potential for bone tissue engineering. Cryogels, mimicking the three-dimensional structure of spongy bone, represent ideal carriers for these cells. We developed poly(2-hydroxyethyl methacrylate) cryogels, containing hydroxyapatite to mimic inorganic bone matrix. Cryogels were additionally supplemented with different types of proteins, namely collagen (Coll), platelet-rich plasma (PRP), immune cells-conditioned medium (CM), and RGD peptides (RGD). The different protein components did not affect scaffolds’ porosity or water-uptake capacity, but altered pore size and stiffness. Stiffness was highest in scaffolds with PRP (82.3 kPa), followed by Coll (55.3 kPa), CM (45.6 kPa), and RGD (32.8 kPa). Scaffolds with PRP, CM, and Coll had the largest pore diameters (~60 µm). Ad-MSCs were osteogenically differentiated on these scaffolds for 14 days. Cell attachment and survival rates were comparable for all four scaffolds. Runx2 and osteocalcin levels only increased in Ad-MSCs on Coll, PRP and CM cryogels. Osterix levels increased slightly in Ad-MSCs differentiated on Coll and PRP cryogels. With differentiation alkaline phosphatase activity decreased under all four conditions. In summary, besides Coll cryogel our PRP cryogel constitutes as an especially suitable carrier for bone tissue engineering. This is of special interest, as this scaffold can be generated with patients’ PRP.
... From a biomechanical perspective, scaffolds should exhibit a low elastic modulus akin to the native tissue while isolating precursor cells from excessive extraneous forces. At the cellular level, adipogenesis is enhanced upon substrates with similar stiffness to the native tissue [5], while decreasing on substrates with higher stiffness [6]. Previous research has mainly focused on design strategies mimicking the mechanical properties of the native tissue as the primary design criteria for soft TECs, while more recently it has been shown that the scaffold should be able to withstand physiological and external loads in an effort to minimize transduction of the applied stress to not only the newly forming tissue, but for much longer periods of time in order to allow tissue maturation and remodelling. ...
... Others include the influence of substrate stiffness and its response to actin organization on differentiation potential. Indeed, a modified substrate enhances the differentiation efficiency of MSCs to specific cell types [3][4][5] . However, studies on remodeling of the nucleus are scarce. ...
Article
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Stem cells undergo drastic morphological alterations during differentiation. While extensive studies have been performed to examine the cytoskeletal remodeling, there is a growing interest to determine the morphological, structural and functional changes of the nucleus. The current study is therefore aimed at quantifying the extent of remodeling of the nuclear morphology of human mesenchymal stem cells during biochemically-induced adipogenic differentiation. Results show the size of nuclei decreased exponentially over time as the lipid accumulation is up-regulated. Increases in the lipid accumulation appear to lag the nuclear reorganization, suggesting the nuclear deformation is a prerequisite to adipocyte maturation. Furthermore, the lamin A/C expression was increased and redistributed to the nuclear periphery along with a subsequent increase in the nuclear aspect ratio. To further assess the role of the nucleus, a nuclear morphology with a high aspect ratio was achieved using microcontact-printed substrate. The cells with an elongated nuclear shape did not efficiently undergo adipogenesis, suggesting the cellular and nuclear processes associated with stem cell differentiation at the early stage of adipogenesis cause a change in the nuclear morphology and cannot be abrogated by the morphological cues. In addition, a novel computational biomechanical model was generated to simulate the nuclear shape change during differentiation and predict the forces acting upon the nucleus. This effort led to the development of computational scaling approach to simulate the experimentally observed adipogenic differentiation processes over 15 days in less than 1.5 hours.
... Pore structure and porosity not only affect cell attachment on the scaffold and cell infiltration into the scaffold but also the nutritional supply and metabolite removal, a factor defined by medium diffusion and characterized by the permeability of the scaffold. The scaffold's stiffness may affect differentiation of the applied progenitor cells-while scaffolds with low stiffness favor adipogenic and chondrogenic differentiation, scaffolds with higher stiffness favor osteogenic differentiation [12]. At the same time scaffolds for bone tissue engineering ideally sustain some flexibility to pass on mechanical stimuli to the cells [13], as this is known to favor osteogenic differentiation [14]. ...
Article
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Cryogels represent ideal carriers for bone tissue engineering. We recently described the osteogenic potential of cryogels with different protein additives, e.g., platelet-rich plasma (PRP). However, these scaffolds raised concerns as different toxic substances are required for their preparation. Therefore, we developed another gelatin (GEL)-based cryogel. This study aimed to compare the two scaffolds regarding their physical characteristics and their influence on osteogenic and osteoclastic cells. Compared to the PRP scaffolds, GEL scaffolds had both larger pores and thicker walls, resulting in a lower connective density. PRP scaffolds, with crystalized calcium phosphates on the surface, were significantly stiffer but less mineralized than GEL scaffolds with hydroxyapatite incorporated within the matrix. The GEL scaffolds favored adherence and proliferation of the osteogenic SCP-1 and SaOS-2 cells. Macrophage colony-stimulating factor (M-CSF) and osteoprotegerin (OPG) levels seemed to be induced by GEL scaffolds. Levels of other osteoblast and osteoclast markers were comparable between the two scaffolds. After 14 days, mineral content and stiffness of the cryogels were increased by SCP-1 and SaOS-2 cells, especially of PRP scaffolds. THP-1 cell-derived osteoclastic cells only reduced mineral content and stiffness of PRP cryogels. In summary, both scaffolds present powerful advantages; however, the possibility to altered mineral content and stiffness may be decisive when it comes to using PRP or GEL scaffolds for bone tissue engineering.
... hASCsfor day 9 were fixed, permeabilized, blocked, and stained with Nile red (Ex: 530/Em: 585 nm), and counterstained with Hoechst (Ex: 460/Em: 490 nm). Phalloidin stains the cytoskeletal/actin while Nile Red acts as marker for the mature adipocytes [43]. The concentration of the dye solution was 1 µg/mL and the incubation time was 15 minutes at room temperature. ...
Preprint
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Stem cell-based therapies carry significant promise for treating human diseases. However, clinical translation of stem cell transplants for effective therapy requires precise non-destructive evaluation of the purity of stem cells with high sensitivity (< 0.001% of the number of cells). Here, we report a novel methodology using hyperspectral imaging (HSI) combined with spectral angle mapping (SAM)-based machine learning analysis to distinguish differentiating human adipose derived stem cells (hASCs) from control stem cells. The spectral signature of adipogenesis generated by the HSI method enabled identification of differentiated cells at single cell resolution. The label-free HSI method was compared with the standard methods such as Oil Red O staining, fluorescence microscopy, and qPCR that are routinely used to evaluate adipogenic differentiation of hASCs. Further, we performed Raman microscopy and multiphoton-based metabolic imaging to provide complimentary information for the functional imaging of the hASCs. Finally, the HSI method was validated using matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging of the stem cells. The study presented here demonstrates that multimodal imaging methods enable label-free identification of stem cell differentiation with high spatial and chemical resolution. This could provide a powerful tool to assess the safety and efficacy of stem cell-based regenerative therapies.
... Similarly soft carriers are thought to induce expression of stem cell markers, e.g. Sox2, in MSCs, which proved to inhibit osteogenic differentiation [212][213][214][215] in favor of adipogenic differentiation [216]. Therefore, carrier stiffness over 60 kPa is proposed to favor osteogenic differentiation of MSCs [217,218]. ...
Article
Today, over 70 diseases and health conditions are known that negatively affect the bone quality directly or indirectly by their medical treatment, establishing the term metabolic bone disease. Already every third hospitalized patient in Europe suffers from musculoskeletal injuries or diseases. Facing an ageing society and a more and more sedentary lifestyle the number of chronic diseases and consequently metabolic bone diseases are expected to continuously increase. In order to investigate the various disease constellations and/or develop new treatment strategies suitable models representing bone metabolism are required. Many in vivo, ex vivo and in vitro models have been described, which have their advantages and limits. We here summarize the advantages and challenges of frequently used models to investigate bone metabolism, focusing on in vitro co-cultures of bone forming osteoblasts and osteoclasts. Comparing own data with published models, we further elaborate the feasibility of commonly used cells lines for such in vitro co-culture models, in order to provide an easy, constantly available, and up-scalable model system for screening alterations in bone metabolism.
... There are studies, showing that carriers with stiffness over 60 kPa favor osteogenic differentiation of MSCs (Engler et al. 2006;Sun et al. 2018). Lowering the stiffness of scaffolds may induce expression of stem cell markers, e.g., Sox2, in MSCs, which proved to inhibit osteogenic differentiation (Ding et al. 2012;Marcellini et al. 2012;Park et al. 2012;Seo et al. 2013) in favor for adipogenic differentiation (Zhao et al. 2014). However, the chosen scaffolds should also not be too stiff to pass on mechanical stimuli to cells (Dawson and Oreffo 2008). ...
Article
Full-text available
Approx. every third hospitalized patient in Europe suffers from musculoskeletal injuries or diseases. Up to 20% of these patients need costly surgical revisions after delayed or impaired fracture healing. Reasons for this are the severity of the trauma, individual factors, e.g, the patients' age, individual lifestyle, chronic diseases, medication, and, over 70 diseases that negatively affect the bone quality. To investigate the various disease constellations and/or develop new treatment strategies, many in vivo, ex vivo, and in vitro models can be applied. Analyzing these various models more closely, it is obvious that many of them have limits and/or restrictions. Undoubtedly, in vivo models most completely represent the biological situation. Besides possible species-specific differences, ethical concerns may question the use of in vivo models especially for large screening approaches. Challenging whether ex vivo or in vitro bone models can be used as an adequate replacement for such screenings, we here summarize the advantages and challenges of frequently used ex vivo and in vitro bone models to study disturbed bone metabolism and fracture healing. Using own examples, we discuss the common challenge of cell-specific normalization of data obtained from more complex in vitro models as one example of the analytical limits which lower the full potential of these complex model systems.
... Second, extensive literature suggested that long chain precursors were more likely to support efficient cell culture 17,19,75 . We wanted a reaction that would be useable with a wide range of materials especially those that were already successful. ...
Thesis
Tailoring hydrogels into biomimetic templates represents a crucial step to build better in-vitro models but it is to date still challenging. Indeed, these synthetic or natural polymeric networks are often so frail they can’t be processed through standard micro-fabrication. Here, we combine a ultra-violet pattern projector with gas permeable microreactors to control gas, reagents and photon distribution and in fine, the reaction kinetics in space and time. Doing so, enabled a generic chemistry that can structure, liquefy or decorate (locally functionalize) common hydrogels. Altogether these three hydrogel engineering operations form a flexible toolbox that supports the most commonly used hydrogels: i.e. Matrigel, Agar-agar, poly(ethylene-glycol) and poly(acryl-amide). We successfully applied this solution to grow cells into standardized micro-niches demonstrating that it can readily address cell culture challenges such has controlled adhesion on topographical structures, standardization of spheroids or culture on shaped Matrigel.
... 35 Shear stress induced by the interstitial level of flow promoted osteogenic differentiation of mesenchymal stem cells (MSCs). 34 Adipogenesis is easily affected by mechanical stimuli such as substrate stiffness, 19,67 compressive forces, 68 and shear stress. 69 PPARγ2 and C/EBPα expressions are shown to be inhibited by fluid shear stress during adipocyte maturation, leading to reduced lipid accumulation. ...
Article
An accurate in vitro model of human adipose tissue could assist in the study of adipocyte function and allow for better tools for screening new therapeutic compounds. Cell culture models on two-dimensional surfaces fall short of mimicking the three-dimensional in vivo adipose environment, while three-dimensional culture models are often unable to support long-term cell culture due, in part, to insufficient mass transport. Microfluidic systems have been explored for adipose tissue models. However, current systems have primarily focused on 2D cultured adipocytes. In this work, a 3D human adipose microtissue was engineered within a microfluidic system. Human adipose-derived stem cells (ADSCs) were used as the cell source for generating differentiated adipocytes. The ADSCs differentiated within the microfluidic system formed a dense lipid-loaded mass with the expression of adipose tissue genetic markers. Engineered adipose tissue showed a decreased adiponectin secretion and increased free fatty acid secretion with increasing shear stress. Adipogenesis markers were downregulated with increasing shear stress. Overall, this microfluidic system enables the on-chip differentiation and development of a functional 3D human adipose microtissue supported by the interstitial flow. This system could potentially serve as a platform for in vitro drug testing for adipose tissue-related diseases.
... By increasing the PEGtriacrylate crosslinker concentration, hyaluronic acid and gelatin hydrogels were fabricated in order to obtain a stiffness range from 20 to 40 0 0 Pa. When hMSCs were cultured on the stiffer hydrogel (40 0 0 Pa) they differentiated into osteogenic lineage whereas adipogenic lineage was favored with the softer hydrogels [31] . Not only does stiffness support stem cell differentiation but also proliferation and self-renewal [32] . ...
Article
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Biomaterial matrices must permit tissue growth and maturation for the success of tissue regeneration strategies. Naturally, this accommodation is achieved via the dynamic remodeling of a cell's extracellular matrix (ECM). Synthetically, hydrolytic or enzymatic degradation are often engineered into materials for this purpose. More recently, supramolecular interactions have been used to provide a biomimetic and tunable mechanism to facilitate tissue formation via their dynamic and reversible non-covalent interactions. By engineering the mechanical and bioactive properties of a material, supramolecular chemists are able to design permissivity into the construct and facilitate tissue integration in-vivo. Furthermore, via the reversibility of non-covalent interactions, injectability and responsiveness can be designed for enhanced delivery and spatio-temporal control. In this review, we delineate the basic considerations needed when designing permissive supramolecular hydrogels for tissue engineering with an eye toward tissue growth and integration. We highlight three archetypal hydrogel systems that have shown well-documented tissue integration in vivo, and provide avenues to assess tissue in-growth. Careful design and assessment of the biomedical potential of a supramolecular hydrogels can inspire the creation of robust and dynamic implants for new tissue engineering applications.
... Likewise, other studies postulate that the differences in cell response are due to mechanical signaling rather than biochemical (Viswanath et al., 2017). The stiffness of substrates also affects stem cell differentiation and proliferation (Engler et al., 2006;Zhao et al., 2014;Gerardo et al., 2019). In sum, both biochemical composition and physical features could play a critical role (Stanton et al., 2019). ...
Article
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Decellularization techniques support the creation of biocompatible extracellular matrix hydrogels, providing tissue-specific environments for both in vitro cell culture and in vivo tissue regeneration. We obtained endometrium derived from porcine decellularized uteri to create endometrial extracellular matrix (EndoECM) hydrogels. After decellularization and detergent removal, we investigated the physicochemical features of the EndoECM, including gelation kinetics, ultrastructure, and proteomic profile. The matrisome showed conservation of structural and tissue-specific components with low amounts of immunoreactive molecules. EndoECM supported in vitro culture of human endometrial cells in two- and three-dimensional conditions and improved proliferation of endometrial stem cells with respect to collagen and Matrigel. Further, we developed a three-dimensional endometrium-like co-culture system of epithelial and stromal cells from different origins. Endometrial co-cultures remained viable and showed significant remodeling. Finally, EndoECM was injected subcutaneously in immunocompetent mice in a preliminary study to test a possible hypoimmunogenic reaction. Biomimetic endometrial milieus offer new strategies in reproductive techniques and endometrial repair and our findings demonstrate that EndoECM has potential for in vitro endometrial culture and as treatment for endometrial pathologies.
... One proof is that mimicking the tendon topography, such as enhancing the substrate modulus, as well as the alignment of type I collagen, promoted the tenogenic differentiation of human MSCs [104]. Some other studies also suggested that adipogenesis and osteogenesis of human MSCs were inclined to occur on substrates with a stiffness close to in vivo microenvironments [105,106]. Considering the native environment that BM-MSCs harbors in bone marrow with sufficient blood supply, whilst NP progenitor cells are in avascular tissue, it is quite understandable that BM-MSCs are more sensitive to serum deprivation than NP progenitor cells; similarly to CEP progenitor cells when compared to NP progenitor cells. ...
Article
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Intervertebral disc (IVD) degeneration occurs with natural ageing and is linked to low back pain, a common disease. As an avascular tissue, the microenvironment inside the IVD is harsh. During degeneration, the condition becomes even more compromised, presenting a significant challenge to the survival and function of the resident cells, as well as to any regeneration attempts using cell implantation. Mesenchymal stem cells (MSCs) have been proposed as a candidate stem cell tool for IVD regeneration. Recently, endogenous IVD progenitor cells have been identified inside the IVD, highlighting their potential for self-repair. IVD progenitor cells have properties similar to MSCs, with minor differences in potency and surface marker expression. Currently, it is unclear how IVD progenitor cells react to microenvironmental factors and in what ways they possibly behave differently to MSCs. Here, we first summarized the microenvironmental factors presented in the IVD and their changes during degeneration. Then, we analyzed the available studies on the responses of IVD progenitor cells and MSCs to these factors, and made comparisons between these two types of cells, when possible, in an attempt to achieve a clear understanding of the characteristics of IVD progenitor cells when compared to MSCs; as well as, to provide possible clues to cell fate after implantation, which may facilitate future manipulation and design of IVD regeneration studies.
... The compressive and tensile strength, and the density and fracture toughness of the graft should be comparable to that of the recipient site. Moreover, it has been shown that the scaffolds' stiffness can have a direct effect on the behaviour of the surrounding cells [138]. Therefore, it becomes critical to choose the material according to the site of procedure and the desired outcome. ...
Article
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Biomimetic materials for hard and soft tissues have advanced in the fields of tissue engineering and regenerative medicine in dentistry. To examine these recent advances, we searched Medline (OVID) with the key terms "biomimetics", "biomaterials", and "biomimicry" combined with MeSH terms for "dentistry" and limited the date of publication between 2010-2020. Over 500 articles were obtained under clinical trials, randomized clinical trials, metanalysis, and systematic reviews developed in the past 10 years in three major areas of dentistry: restorative, orofacial surgery, and periodontics. Clinical studies and systematic reviews along with hand-searched preclinical studies as potential therapies have been included. They support the proof-of-concept that novel treatments are in the pipeline towards groundbreaking clinical therapies for orofacial bone regeneration, tooth regeneration, repair of the oral mucosa, periodontal tissue engineering, and dental implants. Biomimicry enhances the clinical outcomes and calls for an interdisciplinary approach integrating medicine, bioengineering, biotechnology, and computational sciences to advance the current research to clinics. We conclude that dentistry has come a long way apropos of regenerative medicine; still, there are vast avenues to endeavour, seeking inspiration from other facets in biomedical research.
... The stiffness induced behavioral differences among the different kinds of reproducible cells, including dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs) within the dental area [18,19], and others such as valve interstitial cells (VICs) [20] and oral squamous cell carcinoma cells [21] have been investigated, thus bringing more significance to this parameter in tissue engineering and regenerative therapies. It has been established based on the various previous studies on mesenchymal stem cells (MSCs) that the softer substrate matrix is suitable to induce neurogenesis and adipogenesis, while stiffer matrix tended to induce myogenesis chondrogenesis and osteogenesis [14,[22][23][24]. Moreover, as for other cells, upregulated osteogenic markers in DPSCs, PDLSCs, and VICs on stiffer substrates were also reported [18][19][20]. ...
Article
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Recent studies, which aim to optimize maxillary sinus augmentation, have paid significant attention exploring osteogenic potential of maxillary Schneiderian sinus membrane-derived cells (MSSM-derived cells). However, it remains unclear that how MSSM-derived cells could respond to niche’s biomechanical properties. Herein, this study investigated the possible effects of substrate stiffness on rMSSM-derived stem cell fate. Initially, rMSSM-derived stem cells with multiple differentiation potential were successfully obtained. We then fabricated polyacrylamide substrates with varied stiffness ranging from 13 to 68 kPa to modulate the mechanical environment of rMSSM-derived stem cells. A larger cell spreading area and increased proliferation of rMSSM-derived stem cells were found on the stiffer substrates. Similarly, cells became more adhesive as their stiffness increased. Furthermore, the higher stiffness facilitated osteogenic differentiation of rMSSM-derived stem cells. Overall, our results indicated that increase in stiffness could mediate behaviors of rMSSM-derived stem cells, which may serve as a guide in future research to design novel biomaterials for maxillary sinus augmentation.
... Similarly, hydrogel matrix degradation plays an important role in the mechanosensitive differentiation of stem cells . MSCs differentiate into adipogenic and osteogenic lineages when encapsulated into non-degrading hyaluronic acid hydrogels and degradable hydrogels, respectively, regardless of the matrix stiffness (Duarte Campos et al. 2015;Zhao et al. 2014). Khetan et al. demonstrated that matrix degradation is required for tension generation of encapsulated MSCs for osteogenic differentiation (Khetan et al. 2013). ...
Article
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Functional tissue regeneration using synthetic biomaterials requires proliferation and heterotypic differentiation of stem/progenitor cells within a specialized heterogeneous (biophysical–biochemical) microenvironment. The current techniques have limitations to develop synthetic hydrogels, mimicking native extracellular matrix porosity along with heterogeneous microenvironmental cues of matrix mechanics, degradability, microstructure and cell–cell interactions. Here, we have developed a microenvironment modulating system to fabricate in situ porous hydrogel matrix with two or more distinct tailored microenvironmental niches within microbeads and the hydrogel matrix for multicellular tissue regeneration. Electrosprayed pectin-gelatin blended microbeads and crosslinked alginate hydrogel system help to tailor microenvironmental niches of encapsulated cells where two different cells are surrounded by a specific microenvironment. The effect of different microenvironmental parameters associated with the microbead/hydrogel matrix was evaluated using human umbilical-cord mesenchymal stem cells (hUCMSCs). The osteogenic differentiation of hUCMSCs in the hydrogel matrix was evaluated for bone tissue regeneration. This will be the first report on microenvironment modulating microbead-hydrogel system to encapsulate two/more types of cells in a hydrogel, where each cell is surrounded with distinct niches for heterogeneous tissue regeneration.Graphic abstract
... For example, organic-inorganic scaffold holds moderate hydrophilic surfaces (θ: 50 • -60 • ) and high roughness compared to counterpart scaffold, and such properties are crucial to improving cell adhesion, protein adsorption, and cell proliferation than hydrophobic and smooth scaffold surfaces [61]. Organic-inorganic scaffolds hold high elastic modulus, and literature studies reported cell differentiation into neuronal lineage onto soft substrates and an osteogenic lineage on stiffer substrates [62]. The nanocomposite scaffolds acted as biodegradable bioactive scaffolds and could degrade gradually to accelerate the osseointegration process. ...
Article
Bone defects account for a significant proportion of surgeries, increasing healthcare costs and complicating therapies. These factors necessitated cell-instructive bioengineering procedures to support and restore pre-existing bone function. Herein, iron-reinforced hydroxyapatite nanorod was used as an inorganic component, collagen/polycaprolactone was used as an organic component, and tannic acid was used as a surface modifier (onto nanorods), crosslinker, and functional agent. The inorganic–organic nanofibers were coated onto an implant surface using electrospinning. The nanocomposite nanostructural, contact angle, surface chemistries, thermomechanical, physicochemical, cytocompatibility, antioxidant potential, aspects of osseointegration ranging from cell adhesion, proliferation, differentiation, and bone remodeling at bone-implant zone in a rat model were studied. The results support that metal-phenolic networks on nanorods regulate interfacial interactions, nanorods cytocompatibility, antioxidant potential, and phase compatibility between organic and inorganic materials of scaffolds. The organic/inorganic nanocomposite better mimicked the bone matrix and better-supported MC3T3-E1 cell attachment, proliferation, matrix mineralization (intracellular alkaline phosphatase and calcium accumulation), and osteogenic gene expression (OPN, OCN, COL1A2, and RUNX2) to direct cell fate for bone reconstruction than its counterpart organic scaffold. The active bone formation and rebuilding of the bone-implant zone in rat models confirmed the bioactive potential of the studied nanocomposite, and it thus could be used as an interface scaffold to boost osteointegration.
... Cells exhibited spindle-shape morphology on the 0.15 kPa hydrogel, while displaying elongated and cuboidal appearance, similar to osteoblasts on greater stiffness hydrogels. Human MSCs cultured on the 1.5 kPa hydrogel significantly expressed osteopontin, while those cultured on the 4 kPa hydrogel revealed a significant upregulation in the expression of the late osteogenic gene (bone sialoprotein) [174]. ...
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Mesenchymal stem/progenitor cells (MSCs) have a multi-differentiation potential into specialized cell types, with remarkable regenerative and therapeutic results. Several factors could trigger the differentiation of MSCs into specific lineages, among them the biophysical and chemical characteristics of the extracellular matrix (ECM), including its stiffness, composition, topography, and mechanical properties. MSCs can sense and assess the stiffness of extracellular substrates through the process of mechanotransduction. Through this process, the extracellular matrix can govern and direct MSCs’ lineage commitment through complex intracellular pathways. Hence, various biomimetic natural and synthetic polymeric matrices of tunable stiffness were developed and further investigated to mimic the MSCs’ native tissues. Customizing scaffold materials to mimic cells’ natural environment is of utmost importance during the process of tissue engineering. This review aims to highlight the regulatory role of matrix stiffness in directing the osteogenic differentiation of MSCs, addressing how MSCs sense and respond to their ECM, in addition to listing different polymeric biomaterials and methods used to alter their stiffness to dictate MSCs’ differentiation towards the osteogenic lineage.
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The surface structure of biomaterials is of key importance to control its interaction with biological environments. Industrial fabrication and coating processes often introduce particulate nanostructures at implant surfaces. Understanding the cellular interaction with particle-based surface topologies and feature sizes in the colloidal length scale therefore offers the possibility to improve the biological response of synthetic biomaterials. Here, surfaces with controlled topography and regular feature sizes covering the relevant length scale of particulate coatings (100 – 1000 nm) are fabricated by colloidal templating. Using fluorescent microscopy, WST assay and morphology analysis, results show that adhesion and attachment of bone-marrow derived murine stromal cells (ST2) is strongly influenced by the surface feature size while geometric details play an insignificant role. Quantitative analysis shows enhanced cell adhesion, spreading, viability and activity when surface feature size decreases below 200 nm compared to flat surfaces, while larger feature sizes are detrimental to cell adhesion. Kinetic studies reveal that most cells on surfaces with larger features lose contact to the substrate over time. This study identifies colloidal templating as a simple method for creating highly defined model systems to investigate complex cell functions and provides design criteria for the choice of particulate coatings on commercial implant materials.
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Further enhancing the comprehensive properties of hydroxyapatite (HA) bioceramics is still a hot topic in biomedical field. The present study was mainly aimed at investigating the special effect of nanoscale feature on the micromechanical properties and biological performances of HA bioceramics. Three kinds of HA bioceramics with micro‐ to nanocrystalline were fabricated by adopting different sintering technologies, i.e. conventional sintering (CS), two‐step sintering (TSS) and microwave assisted sintering (MAS). The average grain sizes of HA‐CS, HA‐TSS, and HA‐MAS were 412.49 nm, 264.24 nm, and 122.49 nm, respectively. Among them, HA‐MAS nanoceramics had the lowest water contact angle (28.35°) and the highest surface roughness (92.30 nm). Interestingly, the enhanced crack deflection in HA‐MAS transferred from transgranular cracking to intergranular cracking, resulting into its fracture toughness (1.64 MPa m1/2) increased by 84% compared to HA‐CS. Besides, HA‐MAS underwent the elastic‐plastic deformation without pop‐in cracking, and exhibited no indentation size effect (ISE) phenomenon during the nanoindentation tests. The in vitro biological performances showed that HA‐MAS could promote the bone‐like apatite formation and the osteogenic differentiation of BMSCs. In summary, the construction of nanocrystalline is indeed an effective way to simultaneously improve the micromechanical properties and biological performances of HA bioceramics.
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Background and Objectives Photobiomodulation (PBM) describes the influence of light irradiation on biological tissues. Laser light in the near‐infrared (NIR) spectrum has been shown to mitigate pain, reduce inflammation, and promote wound healing. The cellular mechanism that mediates PBM's effects is generally accepted to be at the site of the mitochondria, leading to an increased flux through the electron transport chain and adenosine triphosphate (ATP) production. Moreover, PBM has been demonstrated to reduce oxidative stress through an increased production of reactive oxygen species (ROS)‐sequestering enzymes. The aim of the study is to determine whether these PBM‐induced effects expedite or interfere with the intended stem cell differentiation to the adipogenic lineage. Study Design/Materials and Methods To determine the effects of 1064 nm laser irradiation (fluence of 8.8–26.4 J/cm²) on human mesenchymal stem cells (hMSCs) undergoing adipogenic differentiation, the ATP and ROS levels, and adipogenic markers were quantitatively measured. Results At a low fluence (8.8 J/cm²) the ATP increase was essentially negligible, whereas a higher fluence induced a significant increase. In the laser‐stimulated cells, PBM over time decreased the ROS level compared with the non‐treated control group and significantly reduced the extent of adipogenesis. A reduction in the ROS level was correlated with a diminished lipid accumulation, reduced production of adipose‐specific genetic markers, and delayed the chemically intended adipogenesis. Conclusion We characterized the use of NIR light exposure to modulate adipogenesis. Both the ATP and ROS levels in hMSCs responded to different energy densities. The current study is expected to contribute significantly to the growing field of PBM as well as stem cell tissue engineering by demonstrating the wavelength‐dependent responses of hMSC differentiation. Lasers Surg. Med. © 2020 Wiley Periodicals LLC.
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Various cues from microenvironment that cell lives can regulate cell functions. In addition to biochemical cues, increasing evidence has demonstrated that mechanical cues (namely, stiffness of substrate/matrix in this review) presented by cell microenvironment are also critically important in regulating cell functions. However, most studies on stiffness-regulated cell functions have been mainly focused on 2D condition, which might cannot recapitulate the 3D microenvironment that cell encounters with in vivo. In contrast to 2D microenvironment, researches have already found that cell responds differently to mechanical cues under 3D microenvironment. In this review, the mechanisms of cell mechanosensing and mechanotransduction are briefly presented, followed by an introduction of the most studied 2D/3D platforms. The effects of substrate/matrix stiffness on cell functions, including cell migration, spreading, proliferation, phenotype, and differentiation, under different dimensionalities are summarized and discussed. Finally, the remained questions and future outlooks are also recommended.
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Human adult mesenchymal stem or stromal cells (h-MSC) therapy has gained considerable attention due to the potential to treat or cure diseases given their immunosuppressive properties and tissue regeneration capabilities. Researchers have explored diverse strategies to promote high h-MSC production without losing functional characteristics or properties. Physical stimulus including stiffness, geometry, and topography, chemical stimulus, like varying the surface chemistry, and biochemical stimuli such as cytokines, hormones, small molecules, and herbal extracts have been studied but have yet to be translated to industrial manufacturing practice. In this review, we describe the role of those stimuli on h-MSC manufacturing, and how these stimuli positively promote h-MSC properties, impacting the cell manufacturing field for cell-based therapies. In addition, we discuss other process considerations such as bioreactor design, good manufacturing practice, and the importance of the cell donor and ethics factors for manufacturing potent h-MSC.
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Polysaccharides have been considered as the important linker of organics and minerals in the scaffold for bone repair. In this study, we exploited glycogen as the contactor of collagen and hydroxyapatite to form composite hydrogels, since it could provide stereoscopic multiple sites for reacting with other components. Glycogen was modified with guanido followed by oxidization to aldehyde, which subsequently combined with collagen and hydroxyapatite through Schiff-base bond and electrostatic interaction, respectively. The resulting hydrogels could almost maintain their prototypes within 2 weeks, with little degradation. Their Young's modulus (10 to 70 kPa) and compressive modulus (30 to 432 kPa) were all within the range of properties required for stem cells to differentiate into osteoblasts or cartilage. Additionally, bone mesenchymal stem cells cultured on the composite hydrogels presented desirable cell adhesion, viability and growth. The ratio of collagen/glycogen/hydroxyapatite components could regulate bone mesenchymal stem cell differentiation into osteoblast or chondrocyte, which confirmed by alkaline phosphatase (ALP) activity and related gene expression assays. The results demonstrated that glycogen could be a promising coupling agent for bone scaffold and the C/G/H composite hydrogels would be a potential scaffold for bone tissue engineering.
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Stem cell-based therapies carry significant promise for treating human diseases. However, clinical translation of stem cell transplants for effective treatment requires precise non-destructive evaluation of the purity of stem cells with high sensitivity (<0.001% of the number of cells). Here, a novel methodology using hyperspectral imaging (HSI) combined with spectral angle mapping-based machine learning analysis is reported to distinguish differentiating human adipose-derived stem cells (hASCs) from control stem cells. The spectral signature of adipogenesis generated by the HSI method enables identifying differentiated cells at single-cell resolution. The label-free HSI method is compared with the standard techniques such as Oil Red O staining, fluorescence microscopy, and qPCR that are routinely used to evaluate adipogenic differentiation of hASCs. HSI is successfully used to assess the abundance of adipocytes derived from transplanted cells in a transgenic mice model. Further, Raman microscopy and multiphoton-based metabolic imaging is performed to provide complementary information for the functional imaging of the hASCs. Finally, the HSI method is validated using matrix-assisted laser desorption/ionization-mass spectrometry imaging of the stem cells. The study presented here demonstrates that multimodal imaging methods enable label-free identification of stem cell differentiation with high spatial and chemical resolution.
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Currently there are limited implant-based options for cosmetic breast augmentation, and problems associated with those have been increasingly appreciated, most commonly capsular contracture, which occurs due to a chronic foreign body reaction against non-degradable implant materials such as silicone and polyurethane leading to scar tissue formation, pain, and deformity. The underlying biomechanical concepts with implants create a reciprocal stress-strain relationship with local tissue, whilst acting as a deforming force. This means that with time, as the implant continues to have an effect on surrounding tissue the implant and host's biomechanical properties diverge, making malposition, asymmetry, and other complications more likely. Research directed towards development of alternative therapies based on tissue engineering and regenerative medicine seeks to optimize new tissue formation through modulation of tissue progenitors and facilitating tissue regeneration. Scaffolds can guide the process of new tissue formation by providing both an implant surface and a three-dimensional space that promotes the development of a microenvironment that guides attachment, migration, proliferation, and differentiation of connective tissue progenitors. Important to scaffold design are the architecture, surface chemistry, mechanical properties, and biomaterial used. Scaffolds provide a void in which vascularization, new tissue formation, and remodelling can sequentially occur. They provide a conduit for delivery of the different cell types required for tissue regeneration into a graft site, facilitating their retention and distribution. Whilst recent research from a small number of groups is promising, there are still ongoing challenges to achieving clinical translation. This article summarizes the biomechanical principles of breast implants, how these impact outcomes, and progress in scaffold-guided tissue engineering approaches to cosmetic breast augmentation. LEVEL OF EVIDENCE V: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .
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We have previously demonstrated that the rate of fluid shear stress (ΔSS) can manipulate the fate of mesenchymal stem cells (MSCs) to osteogenic or chondrogenic cells. However, whether ΔSS is comparable to other two means of induction medium and substrate stiffness that have been proven to be potent in differentiation control is unknown. In this study, we subjected MSCs to 1–7 days of osteogenic or chondrogenic chemical induction, or 1–4 days of 37 or 86 kPa of substrate stiffness induction, followed by 20 min of Fast ΔSS (0–0′) or Slow ΔSS (0–2′), which is a laminar FSS that linearly increased from 0 to 10 dyn/cm ² in 0 (Fast) or 2 min (Slow) and maintained at 10 dyn/cm ² for a total of 20 min. We found that 20 min of ΔSS could compete with 5 days' chemical and 2 days' substrate stiffness inductions. Our study confirmed that ΔSS is a powerful tool to control the differentiation of MSCs, which stressed the possible application in MSCs linage specification.
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Spinal cord injury (SCI) is a common, severe damage to the central nervous system. Here, we discuss the use of biomaterials for stem cell transplantation in preclinical and clinical studies for the treatment of patients with SCI, because cell culture materials could influence the differentiation fate of stem cells, and not act only as carriers or scaffolds for delivery of stem cells and their differentiated cells. Therefore, the effects of cell culture materials on stem cell differentiation fate have been discussed. A direct injection of stem cells is the easiest method to transplant stem cells into the site of SCI. However, the stem cell solution tends to leak out from the injection site. Biomaterials such as fibrin have been used to reduce scarring at the transplantation site and facilitate the integration of transplanted stem cells or progenitor cells in animal models of SCI. Transplantation of stem cells using biomaterials (scaffolds or hydrogels) has been reported to be effective for the treatment of SCI in animal models. It would be necessary to investigate the optimal chemical structure, porosity, and morphology of biomaterials used for the transplantation of stem cells.
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Mechanical signalling affects multiple biological processes during development and in adult organisms, including cell fate transitions, cell migration, morphogenesis and immune responses. Here, we review recent insights into the mechanisms and functions of two main routes of mechanical signalling: outside-in mechanical signalling, such as mechanosensing of substrate properties or shear stresses; and mechanical signalling regulated by the physical properties of the cell surface itself. We discuss examples of how these two classes of mechanical signalling regulate stem cell function, as well as developmental processes in vivo. We also discuss how cell surface mechanics affects intracellular signalling and, in turn, how intracellular signalling controls cell surface mechanics, generating feedback into the regulation of mechanosensing. The cooperation between mechanosensing, intracellular signalling and cell surface mechanics has a profound impact on biological processes. We discuss here our understanding of how these three elements interact to regulate stem cell fate and development. Mechanical signalling underlies multiple, fundamental biological processes. Mechanical signals can originate from substrate physical properties or shear stresses, and from changes in the physical properties of the cell surface. The mechanisms underlying these two classes of outside-in signalling and their roles in the regulation of intracellular signalling in cell fate and development are becoming increasingly understood.
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Biophysical cues (especially mechanical cues) embedded in cellular microenvironments show a critical impact on stem cell fate. Despite the capability of traditional hydrogels to mimic the feature of extracellular matrix (ECM) and tune their physicochemical properties via diverse approaches, their relatively large size not only induces biased results, but also hinders high-throughput screening and analysis. In this paper, a microgel model is proposed to recapitulate the role of 3D mechanical microenvironment on stem cell behaviors especially chondrogenesis in vitro. The small diameter of microgels brings the high surface area to volume ratio and then the enlarged diffusion area and shortened diffusion distance of soluble molecules, leading to uniform distribution of nutrients and negligible biochemical gradient inside microgels. To construct ECM-like microenvironment with tunable mechanical strength, three gelatin/hyaluronic acid hybrid microgels with low, medium and high crosslinking densities, i.e., Gel-HA(L), Gel-HA(M) and Gel-HA(H), are fabricated in microfluidic devices by Michael addition reaction between thiolated gelatin (Gel-SH) and ethylsulfated hyaluronic acid (HA-VS) with different substitution degrees of vinyl sulfone groups. Our results show that mouse bone marrow mesenchymal stem cell (BMSC) proliferation, distribution and chondrogenesis are all closely dependent on mechanical microenvironments in microgels. Noteworthily, BMSCs show a clear trend of differentiating into hyaline cartilage in Gel-HA(L) and fibrocartilage in Gel-HA(M) and Gel-HA(H). Whole transcriptome RNA sequencing reveals that mechanical microenvironment of microgels affects BMSC differentiation via TGF-β/Smad signaling pathway, Hippo signaling pathway and Integrin/YAP/TAZ signaling pathway. We believe this microgel model provides a new way to further explore the interaction between cells and 3D microenvironment. Statement of Significance In recent years, hydrogels have been frequently used to construct 3D microenvironment for cells. However, their relatively large size not only brings biased experimental results, but also limits high-throughput screening and analysis. Herein we propose a gelatin/hyaluronic acid microgel model to explore the effects of 3D cellular mechanical microenvironment (biophysical cues) on BMSC behaviors especially chondrogenesis, which can minimize the interference of biochemical gradients. Our results reveal that BMSC differentiation into either hyaline cartilage or fibrocartilage can be regulated via tailoring the mechanical properties of microgels. Whole transcriptome RNA sequencing proves that “TGF-β/Smad signaling pathway”, “Hippo signaling pathway” and “Integrins/YAP/ TAZ signaling pathway” are activated or inhibited in this process.
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Aliphatic polyesters are the synthetic polymers most commonly used in the development of resorbable medical implants/devices. Various three-dimensional (3D) scaffolds have been fabricated from these polymers and used in adipose tissue engineering. However, their systematic evaluation altogether lacks, which makes it difficult to select a suitable degradable polymer to design 3D resorbable implants and/or devices able to effectively mimic the properties of adipose tissue. Additionally, the impact of sterilization methods on the medical devices, if any, must be taken into account. We evaluate and compare five different medical-grade resorbable polyesters with l-lactide content ranging from 50 to 100 mol% and exhibiting different physiochemical properties depending on the comonomer (d-lactide, ε-caprolactone, glycolide, and trimethylene carbonate). The salt-leaching technique was used to prepare 3D microporous scaffolds. A comprehensive assessment of physical, chemical, and mechanical properties of the scaffolds was carried out in PBS at 37 °C. The cell-material interactions and the ability of the scaffolds to promote adipogenesis of human adipose tissue-derived stem cells were assessed in vitro. The diverse physical and mechanical properties of the scaffolds, due to the different composition of the copolymers, influenced human adipose tissue-derived stem cells proliferation and differentiation. Scaffolds made from polymers which were above their glass transition temperature and with low degree of crystallinity showed better proliferation and adipogenic differentiation of stem cells. The effect of sterilization techniques (electron beam and ethylene oxide) on the polymer properties was also evaluated. Results showed that scaffolds sterilized with the ethylene oxide method better retained their physical and chemical properties. Overall, the presented research provides (i) a detailed understanding to select a degradable polymer that has relevant properties to augment adipose tissue regeneration and can be further used to fabricate medical devices/implants; (ii) directions to prefer a sterilization method that does not change polymer properties.
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Mesenchymal stromal cells (MSCs) have gained immense attention over the past two decades due to their multipotent differentiation potential and pro-regenerative and immunomodulatory cytokine secretory profiles. Their ability to modulate the host immune system and promote tolerance has prompted several allogeneic and autologous hMSC-based clinical trials for the treatment of graft-versus-host disease and several other immune-induced disorders. However, clinical success beyond safety is still controversial and highly variable, with inconclusive therapeutic benefits and little mechanistic explanation. This clinical variability has been broadly attributed to inconsistent MSC sourcing, phenotypic characterization, variable potency, and non-standard isolation protocols, leading to functional heterogeneity among administered MSCs. Homogeneous MSC populations are proposed to yield more predictable, reliable biological responses and clinically meaningful properties relevant to cell-based therapies. Limited comparisons of heterogeneous MSCs with homogenous MSCs are reported. This review addresses this gap in the literature with a critical analysis of strategies aimed at decreasing MSC heterogeneity concerning their reported immunomodulatory profiles. Statement of Significance This review collates, summarizes, and critically analyzes published strategies that seek to improve homogeneity in immunomodulatory functioning MSC populations intended as cell therapies to treat immune-based disorders, such as graft-vs-host-disease. No such review for MSC therapies, immunomodulatory profiles and cell heterogeneity analysis is published. Since MSCs represent the most clinically studied experimental cell therapy platform globally for which there remains no US domestic marketing approval, insights into MSC challenges in therapeutic product development are imperative to providing solutions for immunomodulatory variabilities.
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The extracellular matrix (ECM) and its mechanical properties play an important role in regulating the cellular responses that occur during tissue regeneration, wound healing, and disease progression. A growing body of research, especially in the fields of mechanobiology and matrix biology, has been devoted to elucidating how the ECM mechanical environment, both in vitro and in vivo, influences cell fate and function. Synthetic materials that faithfully recapitulate key mechanical properties of native tissues provide an important means to understand the mechanisms by which cells sense and remodel their surrounding mechanical environments. However, tissue mechanics is inherently complex, exhibiting dependencies on many timescales. This review highlights recent progress in synthetic biomaterials, particularly polymer networks that capture critical aspects of the dynamic mechanical properties of soft tissues by exploiting dynamic covalent chemistries. Finally, future directions and opportunities in the development and application of viscoelastic biomaterials are discussed.
Chapter
Systems-wide approaches have gained new insights into mRNA splicing. Improved cryo-EM techniques have elucidated the structure of splice components. Recent findings on the molecular architecture of the human spliceosomeSpliceosome are unveiled. In complement to the normal splicing of introns out of DNA sequences, alternative splicingAlternative splicing is also examined. Many predictions and hypotheses have led to new knowledge about the role of alternative splicingAlternative splicing in the delivery of alterative proteins or peptides, which may be either tissue-specific, developmental stage or disease-specific. Mechanical stimulation of cells can provoke a shift towards a distinct alternative splice variant that may perform a different function, as novel-binding sites are now present or binding sites are accessible due to conformational changes of the protein. The occurrence of alternative splicing in diseases, such as cancer, is being discussed. It is explained how alternative splicingAlternative splicing can be regulated. Finally, the mechanical propertiesMechanical properties of the matrix will be investigated in terms of their effect on the alternative splicingAlternative splicing process and its regulation.
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Hydrogels can be fabricated and designed to exert direct control over stem cells' adhesion and differentiation. In this study, we have investigated the use of polydopamine (pDA)-treatment as a binding platform for bioactive compounds to create a versatile gelatin-alginate (Gel-Alg) hydrogel for tissue engineering applications. Precisely, pDA was used to modify the surface properties of the hydrogel and better control the adhesion and osteogenic differentiation of human adipose-derived stem cells (hASCs). pDA enabled the adsorption of different types of bioactive molecules, including a model osteoinductive drug (dexamethasone) as well as a model pro-angiogenic peptide (QK). The pDA treatment efficiently retained the drug and the peptide compared to the untreated hydrogel and proved to be effective in controlling the morphology, cell area, and osteogenic differentiation of hASCs. Overall, the findings of this study confirm the efficacy of pDA treatment as a valuable strategy to modulate the biological properties of biocompatible Gel-Alg hydrogels and further extend their value in regenerative medicine.
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The osteogenic activity of medical metal can be improved by lowering its surface stiffness and elastic modulus. However, it is very difficult to directly reduce the elastic modulus of medical metal surfaces. In this paper, with selected parameters, the titanium surface was treated via femtosecond laser irradiation. Micro indentation revealed that the femtosecond laser ablation can effectively reduce the surface Young's modulus and Vickers hardness of titanium. Besides, In order to explain the mechanical properties of degradation of titanium surface, Large-scale Atomic/Molecular Massively Parallel Simulator (LAMMPS) was used to simulate the process of laser ablation process of titanium surface, and it was found that after the ablation of titanium surface, voids were produced in the subsurface layer. The simulation showed that the voids are formed by the cavitation of metastable liquid induced by high tensile stress and high temperature during femtosecond laser irradiation. Subsurface voids with a thickness of about 40 nm were observed under the oxide layer in the experiment. Cell experiments showed that the surface with low Young's modulus was more conducive to cell proliferation and osteogenic differentiation.
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The disrupted surface of porous membranes, commonly used in tissue-chip and cellular coculture systems, is known to weaken cell-substrate interactions. Here, we investigated whether disrupted surfaces of membranes with micron and submicron scale pores affect yes-associated protein (YAP) localization and differentiation of adipose-derived stem cells. We found that these substrates reduce YAP nuclear localization through decreased cell spreading, consistent with reduced cell-substrate interactions, and in turn enhance adipogenesis while decreasing osteogenesis.
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Most tissues of the human body are characterized by highly anisotropic physical properties and biological organization. Hydrogels have been proposed as scaffolding materials to construct artificial tissues due to their water-rich composition, biocompatibility, and tunable properties. However, unmodified hydrogels are typically composed of randomly oriented polymer networks, resulting in homogeneous structures with isotropic properties different from those observed in biological systems. Magnetic materials have been proposed as potential agents to provide hydrogels with the anisotropy required for their use on tissue engineering. Moreover, the intrinsic properties of magnetic nanoparticles enable their use as magnetomechanic remote actuators to control the behavior of the cells encapsulated within the hydrogels under the application of external magnetic fields. In this review, we combine a detailed summary of the main strategies to prepare magnetic nanoparticles showing controlled properties with an analysis of the different approaches available to their incorporation into hydrogels. The application of magnetically responsive nanocomposite hydrogels in the engineering of different tissues is also reviewed.
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Objectives Maxillofacial bone defects are the main hindering conditions for traditional dental implant strategies. Guided Bone Regeneration (GBR) is used to handle this situation. The principle of GBR is to use a membrane to prevent the colonization of soft tissue cells of the bone defect and favors the migration of osteogenic linages. Current membranes do not completely fulfill the requirements that an optimal membrane should have, sometimes resulting in non-predictable results. Thus, the need to develop an ideal membrane to perform this duty is clear. Recent developments in bio-manufacturing are driving innovations in membranes technology permitting the active participation of the membrane in the healing and regenerative process trough native tissue mimicking, drug-delivery and cells interaction, away from being a passive barrier. New membranes features need specific evaluation techniques, beyond the International Standard for membrane materials (last reviewed in 2004), being this the rationale for the present review. Nanotechnology application has completely shifted the way of analyzing structural characterization. New progresses on osteoimmmunomodulation have also switched the understanding of cells-membranes interaction. Data and Sources To propose an updated protocol for GBR membranes evaluation, critical reading of the relevant published literature was carried out after a MEDLINE/PubMed database search. Conclusions The main findings are that a potential active membrane should be assessed in its nanostructure, physicochemical and nanomechanical properties, bioactivity and antibacterial, osteoblasts proliferation, differentiation and mineralization. Immunomodulation testing for macrophages recruitment and M2 phenotype promotion in osteoblasts co-culture has to be achieved to completely analyze membranes/tissue interactions.
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There is a critical need to develop in vitro culture systems appropriate for the expansion of adipose tissue to gain new insights into metabolic diseases and to assist in the restoration of tissue defects. Conventional two or three dimensional (2D or 3D) in vitro models of adipocytes require a combination of supplements to induce adipocyte maturation that greatly increases the cost of large-scale industrial production. In the present study, a microporous, perforated bacterial cellulose (BC)-assisted culture system was developed that promoted the adhesion, proliferation, and adipogenic differentiation of preadipocytes. Additionally, the system maintained the cells as mature unilocular adipocytes ex vivo in normal cell culture medium in long-term culture. All cells were derived from isolated adipose tissue without the use of expensive enzymes for tissue digestion. In contrast to culture in hard tissue culture plates, preadipocytes in the soft 3D environments formed multidimensional interlaced cell contacts, undergoing significant spontaneous lipid accumulation and could be cultured for up to 3 months in maintenance medium. More importantly, the cultured adipose tissue-derived cell bank created here was able to produce injury repair activators that promoted the proliferation of fibroblasts with little fibrosis and the functional differentiation of myoblasts, displaying the potential for use in adipose reconstruction. Thus, the present study demonstrated the potential of a mechanically-flexible BC scaffold to generate volume tunable adipose constructs and provides a low-cost and user-friendly strategy for large-scale industrial production of adipose tissue.
Chapter
One of the most-used man-made materials is concrete, a mixture of cement, sand, aggregates, water, and admixtures. It can be seen everywhere: in tunnels, bridges, and high-rise buildings. Ever since concrete was rediscovered two centuries ago, it has been studied in detail in order to optimize the material and to solve its inherent problems. Most people know that concrete is gray, hard, and strong, and expect it to last decades and even centuries. Unfortunately, this is not always the case. Concrete is a material which can cope with high compressive forces but when it is subjected to tensile forces, it may crack. This cracking is based on several environmental and loading conditions, but the fact that concrete is prone to crack is a big issue. When cracking occurs and potentially harmful substances enter the interior of concrete, the concrete matrix may be damaged and even be destroyed. That is the reason why a lot of maintenance and repair works are due in order to increase the durability and lifetime of structures in civil engineering. One way of dealing with these issues is the modification of the material itself, making it less prone to cracking and the durability-related consequences. An example is the use of reinforcements, coping with the tensile forces when concrete cracks. Cracks are not harmful but intruding substances may trigger the corrosion of the iron rebars leading to structural failure, which is again unwanted. In consequence, along the history, different materials were investigated and added to concrete to solve the previous adverted problems. So, why not try adding the white powder superabsorbent polymers in the cementitious material in order to solve these issues?
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The aim of this study is to investigate polyacrylamide‐based hydrogels stress relaxation and the subsequent impact on the osteogenic differentiation of human mesenchymal stem cells (hMSCs). Different hydrogels are synthesized by varying the amount of cross‐linker and the ratio between the monomers (acrylamide and acrylic acid), and characterized by compression tests. It has been found that hydrogels containing 18% of acrylic acid exhibit an average relaxation of 70%, while pure polyacrylamide gels show an average relaxation of 15%. Subsequently, hMSCs are cultured on two different hydrogels functionalized with a mimetic peptide of the bone morphogenetic protein‐2 to enable cell adhesion and favor their osteogenic differentiation. Phalloidin staining shows that for a constant stiffness of 55 kPa, a hydrogel with a low relaxation (15%) leads to star‐shaped cells, which is typical of osteocytes, while a hydrogel with a high relaxation (70%) presents cells with a polygonal shape characteristic of osteoblasts. Immunofluorescence labeling of E11, strongly expressed in early osteocytes, also shows a dramatically higher expression for cells cultured on the hydrogel with low relaxation (15%). These results clearly demonstrate that, by fine‐tuning hydrogels stress relaxation, hMSCs differentiation can be directed toward osteoblasts, and even osteocytes, which is particularly rare in vitro. Poly(acrylamide‐co‐acrylic acid) hydrogels are synthesized with controlled stiffness, ranging between 5 and 145 kPa. Hydrogels containing 0% or 18% of acrylic acid exhibit an average relaxation of 15% and 70%, respectively. Hydrogels with the same stiffness (55 kPa) and different stress relaxation (70% and 15%) induce mesenchymal stem cells differentiation into osteoblasts and osteocytes, respectively.
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Biomolecular simulations are computationally expensive. Simulating time histories larger than seconds remain elusive even with the help of supercomputers. Biological phenomena are multiscale in nature. The dynamics range from atomistic to microscale. Herein a recently developed scaling approach, based on the method of multiple scales, is used to accomplish a long term simulation of a subcellular system. The first key advantage of this approach is the drastic reduction in computational time. This approach is illustrated using a mesenchymal stem cell as it undergoes adipogenic differentiation, a process that takes 15 days, which was simulated in less than 1.5 hours on a typical desktop computer. The second key advantage of the high-speed simulation is that it facilitates the study of mechanical properties, such as nucleus membrane stiffness, that are difficult to measure experimentally with certainty.
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Many fundamental cell processes, such as angiogenesis, neurogenesis and cancer metastasis, are thought to be modulated by extracellular matrix stiffness. Thus, the availability of matrix substrates having well-defined stiffness profiles can be of great importance in biophysical studies of cell-substrate interaction. Here, we present a method to fabricate biocompatible hydrogels with a well defined and linear stiffness gradient. This method, involving the photopolymerization of films by progressively uncovering an acrylamide/bis-acrylamide solution initially covered with an opaque mask, can be easily implemented with common lab equipment. It produces linear stiffness gradients of at least 115 kPa/mm, extending from ,1 kPa to 240 kPa (in units of Young's modulus). Hydrogels with less steep gradients and narrower stiffness ranges can easily be produced. The hydrogels can be covalently functionalized with uniform coatings of proteins that promote cell adhesion. Cell spreading on these hydrogels linearly correlates with hydrogel stiffness, indicating that this technique effectively modifies the mechanical environment of living cells. This technique provides a simple approach that produces steeper gradients, wider rigidity ranges, and more accurate profiles than current methods. This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding:
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MSCs are nonhematopoietic stromal cells that are capable of differentiating into, and contribute to the regeneration of, mesenchymal tissues such as bone, cartilage, muscle, ligament, tendon, and adipose. MSCs are rare in bone marrow, representing ∼1 in 10,000 nucleated cells. Although not immortal, they have the ability to expand manyfold in culture while retaining their growth and multilineage potential. MSCs are identified by the expression of many molecules including CD105 (SH2) and CD73 (SH3/4) and are negative for the hematopoietic markers CD34, CD45, and CD14. The properties of MSCs make these cells potentially ideal candidates for tissue engineering. It has been shown that MSCs, when transplanted systemically, are able to migrate to sites of injury in animals, suggesting that MSCs possess migratory capacity. However, the mechanisms underlying the migration of these cells remain unclear. Chemokine receptors and their ligands and adhesion molecules play an important role in tissue-specific homing of leukocytes and have also been implicated in trafficking of hematopoietic precursors into and through tissue. Several studies have reported the functional expression of various chemokine receptors and adhesion molecules on human MSCs. Harnessing the migratory potential of MSCs by modulating their chemokine-chemokine receptor interactions may be a powerful way to increase their ability to correct inherited disorders of mesenchymal tissues or facilitate tissue repair in vivo. The current review describes what is known about MSCs and their capacity to home to tissues together with the associated molecular mechanisms involving chemokine receptors and adhesion molecules.Disclosure of potential conflicts of interest is found at the end of this article.
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Human mesenchymal stem cells (hMSCs) have been shown to trans-differentiate into neuronal-like cells by culture in neuronal induction media, although the mechanism is not well understood. Topography can also influence cellular responses including enhanced differentiation of progenitor cells. As extracellular matrix (ECM) in vivo comprises topography in the nanoscale, we hypothesize that nanotopography could influence stem cell differentiation into specific non-default pathways, such as transdifferentiation of hMSCs. Differentiation and proliferation of hMSCs were studied on nanogratings of 350 nm width. Cytoskeleton and nuclei of hMSCs were aligned and elongated along the nanogratings. Gene profiling and immunostaining showed significant up-regulation of neuronal markers such as microtubule-associated protein 2 (MAP2) compared to unpatterned and micropatterned controls. The combination of nanotopography and biochemical cues such as retinoic acid further enhanced the up-regulation of neuronal marker expressions, but nanotopography showed a stronger effect compared to retinoic acid alone on unpatterned surface. This study demonstrated the significance of nanotopography in directing differentiation of adult stem cells.
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Stem cell fate is influenced by a number of factors and interactions that require robust control for safe and effective regeneration of functional tissue. Coordinated interactions with soluble factors, other cells, and extracellular matrices define a local biochemical and mechanical niche with complex and dynamic regulation that stem cells sense. Decellularized tissue matrices and synthetic polymer niches are being used in the clinic, and they are also beginning to clarify fundamental aspects of how stem cells contribute to homeostasis and repair, for example, at sites of fibrosis. Multifaceted technologies are increasingly required to produce and interrogate cells ex vivo, to build predictive models, and, ultimately, to enhance stem cell integration in vivo for therapeutic benefit.
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Recent studies have demonstrated the existence of a subset of cells in human bone marrow capable of differentiating along multiple mesenchymal lineages. Not only do these mesenchymal stem cells (MSCs) possess multilineage developmental potential, but they may be cultured ex vivo for many passages without overt expression of a differentiated phenotype. The goals of the current study were to determine the growth kinetics, self-renewing capacity, and the osteogenic potential of purified MSCs during extensive subcultivation and following cryopreservation. Primary cultures of MSCs were established from normal iliac crest bone marrow aspirates, an aliquot was cryopreserved and thawed, and then both frozen and unfrozen populations were subcultivated in parallel for as many as 15 passages. Cells derived from each passage were assayed for their kinetics of growth and their osteogenic potential in response to an osteoinductive medium containing dexamethasone. Spindle-shaped human MSCs in primary culture exhibit a lag phase of growth, followed by a log phase, finally resulting in a growth plateau state. Passaged cultures proceed through the same stages, however, the rate of growth in log phase and the final number of cells after a fixed period in culture diminishes as a function of continued passaging. The average number of population doublings for marrow-derived adult human MSCs was determined to be 38 ± 4, at which time the cells finally became very broad and flattened before degenerating. The osteogenic potential of cells was conserved throughout every passage as evidenced by the significant increase in APase activity and formation of mineralized nodular aggregates. Furthermore, the process of cryopreserving and thawing the cells had no effect on either their growth or osteogenic differentiation. Importantly, these studies demonstrate that replicative senescence of MSCs is not a state of terminal differentiation since these cells remain capable of progressing through the osteogenic lineage. The use of population doubling potential as a measure of biological age suggests that MSCs are intermediately between embryonic and adult tissues, and as such, may provide an in situ source for mesenchymal progenitor cells throughout an adult's lifetime. J. Cell. Biochem. 64:278–294. © 1997 Wiley-Liss, Inc.
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Several experimental findings that are inconsistent with the view that the spleen colony-forming cell (CFU-S) is the primary haemopoietic stem cell are reviewed. Recovery of CFU-S, both quantitatively and qualitatively, can proceed differently depending upon the cytotoxic agent or regime used to bring about the depletion. The virtual immortality of the stem cell population is at variance with evidence that the CFU-S population has an 'age-structure' which has been invoked by several workers to explain experimental and clinical observations. To account for these inconsistencies, a hypothesis is proposed in which the stem cell is seen in association with other cells which determine its behaviour. It becomes essentially a fixed tissue cell. Its maturation is prevented and, as a result, its continued proliferation as a stem cell is assured. Its progeny, unless they can occupy a similar stem cell 'niche', are first generation colony-forming cells, which proliferate and mature to acquire a high probability of differentiation, i.e., they have an age-structure. Some of the experimental situations reviewed are discussed in relation to the proposed hypothesis.
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The differentiation of adipocytic and osteogenic cells has been investigated in cultures of adult rat marrow stromal cells. Adipocytic differentiation was assessed using morphological criteria, changes in expression of procollagen mRNAs, consistent with a switch from the synthesis of predominantly fibrillar (types I and III) to basement membrane (type IV) collagen, and the induction of expression of aP2, a specific marker for differentiation of adipocytes. Osteogenic differentiation was assessed on the basis of changes in the abundance of the mRNAs for the bone/liver/kidney isozyme of alkaline phosphatase and the induction of mRNAs for bone sialoprotein and osteocalcin. In the presence of foetal calf serum and dexamethasone (10(-8) M) there was always differentiation of both adipocytic and osteogenic cells. When the steroid was present throughout primary and secondary culture the differentiation of osteogenic cells predominated. Conversely, when dexamethasone was present in secondary culture only, the differentiation of adipocytes predominated. When marrow stromal cells were cultured in the presence of dexamethasone in primary culture and dexamethasone and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3; 10(-8) M) in secondary culture, the differentiation of adipocytes was inhibited whereas the differentiation of osteogenic cells was enhanced, as assessed by an increase in expression of osteocalcin mRNA. The results, therefore, demonstrate an inverse relationship between the differentiation of adipocytic and osteogenic cells in this culture system and are consistent with the possibility that the regulation of adipogenesis and osteogenesis can occur at the level of a common precursor in vivo.
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The bone morphogenetic proteins were originally identified based on their ability to induce ectopic bone formation in vivo and have since been identified as members of the transforming growth factor-beta gene superfamily. It has been well established that the bone morphogenetic cytokines enhance osteogenic activity in bone marrow stromal cells in vitro. Recent reports have described how bone morphogenetic proteins inhibited myogenic differentiation of bone marrow stromal cells in vitro. In vivo, bone marrow stromal cells differentiate along the related adipogenic pathway with advancing age. The current work reports the inhibitory effects of the bone morphorphogenetic proteins on adipogenesis in a multipotent murine bone marrow stromal cell line, BMS2. When exposed to bone morphogenetic protein-2, the pre-adipocyte BMS2 cells exhibited the expected induction of the osteogenic-related enzyme, alkaline phosphatase. Following induction of the BMS2 cells with adipogenic agonists, adipocyte differentiation was assessed by morphologic, enzymatic, and mRNA markers. Flow cytometric analysis combined with staining by the lipophilic fluorescent dye, Nile red, was used to quantitate the extent of lipid accumulation within the BMS2 cells. By this morphologic criteria, the bone morphogenetic proteins inhibited adipogenesis at concentrations of 50 to 500 ng/ml. This correlated with decreased levels of adipocyte specific enzymes and mRNAs. The BMS2 pre-adipocytes constitutively expressed mRNA encoding bone morphogenetic protein-4 and this was inhibited by adipogenic agonists. Together, these findings demonstrate that bone morphogenetic proteins act as adipogenic antagonists. This supports the hypothesis that adipogenesis and osteogenesis in the bone marrow microenvironment are reciprocally regulated.
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A growing body of data suggests that the bone marrow stroma contains a population of pluripotent cells capable of differentiating into adipocytes, osteoblasts, and lymphohematopoietic supporting cells. In this work, the murine stromal cell lines BMS2 and +/+ 2.4 have been examined as preadipocytes and adipocytes for evidence of osteoblastic gene expression. Adipocyte differentiation has been quantitated using fluorescence activated cell sorting. Within 7-10 days of adipocyte induction by treatment with glucocorticoids, indomethacin, and methylisobutylxanthine, between 40% to 50% of the cells contain lipid vacuoles and exhibit a characteristic adipocyte morphology. Based on immunocytochemistry, both the adipocytes and preadipocytes express a number of osteoblastic markers; these include alkaline phosphatase, osteopontin, collagen (I, III), bone sialoprotein II, and fibronectin. Based on biochemical assays, the level of alkaline phosphatase expression is not significantly different between preadipocyte and adipocyte cells. However, unlike rat cell lines, dexamethasone exposure causes a dose-dependent decrease in enzyme activity. The steady-state mRNA levels of the osteoblast associated genes varies during the process of adiopogenesis. The relative level of collagen I and collagen III mRNA is lower in adipocyte-induced cells when compared to the uninduced controls. Osteocalcin mRNA is detected in preadipocytes but absent in adipocytes. These data indicate that osteoblastic gene expression is detected in cells capable of undergoing adipocyte differentiation, consistent with the hypothesis that these cell lineages are interrelated.
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Recent studies have demonstrated the existence of a subset of cells in human bone marrow capable of differentiating along multiple mesenchymal lineages. Not only do these mesenchymal stem cells (MSCs) possess multilineage developmental potential, but they may be cultured ex vivo for many passages without overt expression of a differentiated phenotype. The goals of the current study were to determine the growth kinetics, self-renewing capacity and the osteogenic potential of purified MSCs during extensive subcultivation and following cryopreservation. Primary cultures of MSCs were established from normal iliac crest bone marrow aspirates, an aliquot was cryopreserved and thawed, and then both frozen and unfrozen populations were subcultivated in parallel for as many as 15 passages. Cells derived from each passage were assayed for their kinetics of growth and their osteogenic potential in response to an osteoinductive medium containing dexamethasone. Spindle-shaped human MSCs in primary culture exhibit a lag phase of growth, followed by a log phase, finally resulting in a growth plateau state. Passaged cultures proceed through the same stages, however, the rate of growth in log phase and the final number of cells after a fixed period in culture diminishes as a function of continued passaging. The average number of population doublings for marrow-derived adult human MSCs was determined to be 38 +/- 4, at which time the cells finally became very broad and flattened before degenerating. The osteogenic potential of cells was conserved throughout every passage as evidenced by the significant increase in APase activity and formation of mineralized nodular aggregates. Furthermore, the process of cryopreserving and thawing the cells had no effect on either their growth or osteogenic differentiation. Importantly, these studies demonstrate that replicative senescence of MSCs is not a state of terminal differentiation since these cells remain capable of progressing through the osteogenic lineage. The use of population doubling potential as a measure of biological age suggests that MSCs are intermediately between embryonic and adult tissues, and as such, may provide an in situ source for mesenchymal progenitor cells throughout an adult's lifetime.
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Transforming growth factor (TGF)-beta-induced chondrogenesis of mesenchymal stem cells derived from bone marrow involves the rapid deposition of a cartilage-specific extracellular matrix. The sequential events in this pathway leading from the undifferentiated stem cell to a mature chondrocyte were investigated by analysis of key matrix elements. Differentiation was rapidly induced in cells cultured in the presence of TGF-beta 3 or -beta 2 and was accompanied by the early expression of fibromodulin and cartilage oligomeric matrix protein. An increase in aggrecan and versican core protein synthesis defined an intermediate stage, which also involved the small leucine-rich proteoglycans decorin and biglycan. This was followed by the appearance of type II collagen and chondroadherin. The pathway was also characterized by the appearance of type X collagen, usually associated with hypertrophic cartilage. There was also a change in the pattern of sulfation of chondroitin sulfate, with a progressive increase in the proportion of 6-sulfated species. The major proportion of newly synthesized glycosaminoglycan was part of an aggregating proteoglycan network. These data allow us to define the phenotype of the differentiated cell and to understand in greater detail the sequential process of matrix assembly.
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This study evaluated chondrogenesis of mesenchymal progenitor stem cells (MSCs) cultured initially under pre-confluent monolayer conditions exposed to transforming growth factor-beta1 (TGF-beta1), and subsequently in three-dimensional cultures containing insulin-like growth factor I (IGF-I). Bone marrow aspirates and chondrocytes were obtained from horses and cultured in monolayer with 0 or 5 ng of TGF-beta 1 per ml of medium for 6 days. TGF-beta 1 treated and untreated cultures were distributed to three-dimensional fibrin disks containing 0 or 100 ng of IGF-I per ml of medium to establish four treatment groups. After 13 days, cultures were assessed by toluidine blue staining, collagen types I and II in situ hybridization and immunohistochemistry, proteoglycan production by [35S]-sulfate incorporation, and disk DNA content by fluorometry. Mesenchymal cells in monolayer cultures treated with TGF-beta1 actively proliferated for the first 4 days, developed cellular rounding, and formed cell clusters. Treated MSC cultures had a two-fold increase in medium proteoglycan content. Pretreatment of MSCs with TGF-beta1 followed by exposure of cells to IGF-I in three-dimensional culture significantly increased the formation of markers of chondrocytic function including disk proteoglycan content and procollagen type II mRNA production. However, proteoglycan and procollagen type II production by MSC's remained lower than parallel chondrocyte cultures. MSC pretreatment with TGF-beta1 without sequential IGF-I was less effective in initiating expression of markers of chondrogenesis. This study indicates that although MSC differentiation was less than complete when compared to mature chondrocytes, chondrogenesis was observed in IGF-I supplemented cultures, particularly when used in concert with TGF-beta1 pretreatment.
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A number of adult mesenchymal tissues contain subpopulations of undifferentiated cells, which retain the capacity to differentiate along multiple lineages. These mesenchymal progenitor cells may be cultured in an undifferentiated state and, when given the appropriate signals, differentiate into an expanding list of several mesenchymal and a few ectodermal derived tissues. The maintenance and propagation of the multipotential nature of these progenitor cell populations are crucially dependent on the isolation protocol, the culture expansion conditions, particularly the properties of the fetal bovine serum supplement in the culture medium. This article describes a method for selection of the appropriate serum lot, and introduces a simplified isolation technique to optimize the yield of progenitor cells that maintain the capability of undergoing multilineage differentiation in response to appropriate cues. Cell populations isolated and culture expanded in this manner, by virtue of their multiple differentiation potential, should serve as ideal candidate cells for tissue engineering applications for the repair and regeneration of tissue damaged by disease and or trauma.
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We describe the development of an injectable, cell-containing hydrogel that supports cell proliferation and growth to permit in vivo engineering of new tissues. Two thiolated hyaluronan (HA) derivatives were coupled to four alpha,beta-unsaturated ester and amide derivatives of poly(ethylene glycol) (PEG) 3400. The relative chemical reactivity with cysteine decreased in the order PEG-diacrylate (PEGDA)>PEG-dimethacrylate>PEG-diacrylamide>PEG-dimethacrylamide. The 3-thiopropanoyl hydrazide derivative (HA-DTPH) was more reactive than the 4-thiobutanoyl hydrazide, HA-DTBH. The crosslinking of HA-DTPH with PEGDA in a molar ratio of 2:1 occurred in approximately 9 min, suitable for an in situ crosslinking applications. The in vitro cytocompatibility and in vivo biocompatibility were evaluated using T31 human tracheal scar fibroblasts, which were suspended in medium in HA-DTPH prior to addition of the PEGDA solution. The majority of cells survived crosslinking and the cell density increased tenfold during the 4-week culture period in vitro. Cell-loaded hydrogels were also implanted subcutaneously in the flanks of nude mice, and after immunohistochemistry showed that the encapsulated cells retained the fibroblast phenotype and secreted extracellular matrix in vivo. These results confirm the potential utility of the HA-DTPH-PEGDA hydrogel as an in situ crosslinkable, injectable material for tissue engineering.
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During prolonged cultivation ex vivo, adult bone marrow stromal stem cells (BMSCs) undergo two probably interdependent processes, replicative aging and a decline in differentiation potential. Recently, our results with primary human fibroblasts indicated that growth on denatured collagen (DC) matrix results in the reduction of the rate of cellular aging. The present study has been undertaken to test whether the growth of human BMSCs under the same conditions would translate into preservation of cellular aging-attenuated functions, such as the ability to express HSP70 in response to stress as well as of osteogenic differentiation potential. We report here that growth of BMSCs on a DC matrix versus tissue culture polystyrene significantly reduced one of the main manifestations of cellular aging, the attenuation of the ability to express a major protective stress response component, HSP70, increased the proliferation capacity of ex vivo expanded BMSCs, reduced the rate of morphological changes, and resulted in a dramatic increase in the retention of the potential to express osteogenic-specific functions and markers upon treatment with osteogenic stimulants. BMSCs are a promising and increasingly important cell source for tissue engineering as well as cell and gene therapeutic strategies. For use of BMSCs in these applications, ex vivo expansion is necessary to obtain a sufficient, therapeutically useful, number of cells; however, this results in the loss of differentiation potential. This problem is especially acute in older patients where more extensive in vitro expansion of smaller number of stem/progenitor cells is needed. The finding that growth on certain biomaterials preserves aging-attenuated functions, enhances proliferation capacity, and maintains differentiation potential of BMSCs indicates a promising approach to address this problem.
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Cells sense and respond to mechanical force. However, the mechanisms of transduction of extracellular matrix (ECM) forces to biochemical signals are not known. After removing the cell membrane and soluble proteins by Triton X-100 extraction, we found that the remaining complex (Triton cytoskeletons) activated Rap1 upon stretch. Rap1 guanine nucleotide exchange factor, C3G, was required for this activation; C3G as well as the adaptor protein, CrkII, in cell extract bound to Triton cytoskeletons in a stretch-dependent manner. CrkII binding, which was Cas dependent, correlated with stretch-dependent tyrosine phosphorylation of proteins in Triton cytoskeletons including Cas at the contacts with ECM. These in vitro findings were compatible with in vivo observations of stretch-enhanced phosphotyrosine signals, accumulation of CrkII at cell-ECM contacts, and CrkII-Cas colocalization. We suggest that mechanical force on Triton cytoskeletons activates local tyrosine phosphorylation, which provides docking sites for cytosolic proteins, and initiates signaling to activate Rap1.
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Recently, cell-based approaches utilizing adipogenic progenitor cells for fat tissue engineering have been developed and reported to have success in promoting in vivo adipogenesis and the repair of defect sites. For autologous applications, human bone marrow-derived mesenchymal stem cells (MSCs) have been suggested as a potential cell source for adipose tissue engineering applications due to their ability to be isolated and ex vivo expanded from adult bone marrow aspirates and their versatility for pluripotent differentiation into various mesenchymal lineages including adipogenic. Due to the relatively low frequency of MSCs present within bone marrow, extensive ex vivo expansion of these cells is necessary to obtain therapeutic cell populations for tissue engineering strategies. Currently, utilization of MSCs for adipose tissue engineering is limited due to the attenuation of their adipogenic differentiation potential following extensive ex vivo expansion on conventional tissue culture plastic (TCP) substrates. In the present study, the ability of a denatured collagen type I (DC) matrix to preserve MSC adipogenic potential during ex vivo expansion was examined. Adipocyte-related markers and functions were examined in vitro in response to adipogenic culture conditions for 21 days in comparison to early passage MSCs and late passage MSCs ex vivo expanded on TCP. The results demonstrated significant preservation of the ability of late passage MSCs ex vivo expanded on the DC matrix to express adipogenic markers (fatty acid-binding protein-4, lipoprotein lipase, acyl-CoA synthetase, adipsin, facilitative glucose transporter-4, and accumulation of lipids) similar to the early passage cells and in contrast to late passage MSCs expanded on TCP. The ability of the DC matrix to preserve adipocyte-related markers and functions of MSCs following extensive ex vivo expansion represents a novel culture technique to expand functional adipogenic progenitors for tissue engineering applications.