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Mesenchymal Stem Cells for Optimizing Bone Volume at the Dental Implant Recipient Site

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Chapter 9
Mesenchymal Stem Cells for Optimizing Bone Volume
at the Dental Implant Recipient Site
Mustafa Ayna, Aydin Gülses, Jörg Wiltfang and
Yahya Açil
Additional information is available at the end of the chapter
http://dx.doi.org/10.5772/intechopen.68514
Provisional chapter
© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,
and reproduction in any medium, provided the original work is properly cited.
DOI: 10.5772/intechopen.68514
Mesenchymal Stem Cells for Optimizing Bone Volume
at the Dental Implant Recipient Site
Mustafa Ayna, Aydin Gülses,
Jörg Wiltfang and Yahya Açil
Additional information is available at the end of the chapter
Abstract
Inadequate bone volume at the implant recipient site presents a clinical challenge for
many dental practitioners. To overcome these problems, several approaches have been
developed and are currently used, including bone grafting strategies and distraction
osteogenesis. Mesenchymal stem cells (MSCs) have gained their popularity within the last
two decades, with regard to promising clinical results in improving the bone architecture
at the implant recipient site. The aim of this chapter was to briey outline the accessibility
properties, dierentiation capacities, isolation, and characterization of MSCs with regard
to optimizing bone volume in dental implantology. Additionally, potential benets and
pitfalls are discussed in comparison with the conventional bone augmentation techniques.
Keywords: bone, dental, implantology, mesenchymal stem cells, platelet-rich plasma
1. Introduction
Dental implant therapies became an integral part of the daily dental practice. The success rate
of implants is related to the correct position and angulation of implants in residual crest, so
that height and thickness of bone augmentation can allow predictable results [1]. Therefore,
the qualitative and quantitative characteristics of the surrounding tissues at the implant recip-
ient site play a key role in the success of the procedure. Systemic diseases such as osteoporo-
sis, changes in vitamin D metabolism, diabetes and adverse pregnancy outcomes, and local
factors such as periodontitis, infections, pre-existing cysts or tumors, and traumatic extrac-
tions might result in the loss of both alveolar bone volume and quality and complicates the
feasibility and long-term clinical outcomes of dental implant rehabilitation.
© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.
Various reconstructive surgical interventions could be necessary to regenerate bone defects
prior to implant placement. In the literature, there are numerous clinical and experimen-
tal studies presenting techniques with dierent results that overcome the problems related
to the insucient bone volume at the edentulous alveolar ridge. Among these, the mostly
performed surgical procedures to obtain bone augmentation are guided bone regeneration
techniques via synthetic materials, xenografts or allografts, distraction osteogenesis of the
alveolus, and the augmentation with autogenous bone blocks, which is thought to be the gold
standard to obtain accurate bone volume and morphology with long-term predictable results.
All techniques described above have their own advantages and pitfalls.
2. Conventional bone grafting strategies
2.1. Synthetic bone graft materials
A variety of articial bone substitutes were used to reconstruct bone defects of the jaws.
Synthetic bone grafts at most possess only osseoconductive characteristics and ideally should
be biocompatible, show minimal brotic reaction, undergo remodeling, support new bone
formation, and should have a similar strength or similar mechanical characteristics to that of
the cortical/cancellous bone being replaced [2]. Availability of synthetic bone graft materials
would eliminate the need for invasive graft-harvesting procedure and the dangers of patho-
gen transmission from immunogenic reaction to bank bone [2, 3]. In the maxillofacial recon-
struction, the mostly used synthetic bone graft materials are:
• calcium phosphates,
• calcium phosphate cements,
• beta-tricalcium phosphates,
• synthetic hydroxyapatites,
• coralline hydroxyapatites, and
• bioactive glasses.
It is obvious that synthetic bone substitutes only have osteoconductive properties, and there is
a need for improvement in their mechanical and degradation properties to ensure the replace-
ment of the graft material with the living bone.
2.2. Allografts
The term allograft describes transplants between two subjects of the same species. Complications
associated with the harvesting of autogenous bone have led to gain in their popularity as a
treatment option in maxillofacial reconstruction. Allografts might oer the same characteris-
tics as autograft; however, they do not present same osteogenic cells and therefore fulll only
the demand of osteoconductivity and serve mostly as a scaold for new bone formation [2].
Mesenchymal Stem Cells - Isolation, Characterization and Applications186
The advantages of allografts include availability and avoidance of morbidity associated with
autogenous bone graft harvesting.
It is obvious that tissue safety is a major concern in transplantation. The major risk and
disadvantage related to the use of allografts are the transmission of infectious agents from
donor to recipient, which could result in microbial contamination from an infected donor,
during collection of the tissue from donors or the environment and during processing of the
tissues [4].
Viral transmission is a potential risk that is historically and serologically reported in asso-
ciation with allografts. Despite the exceedingly low risk, the transmission of human immu-
nodeciency virus (HIV-1) from seronegative cadaveric donors has reported in Refs. [4, 5].
During the history of allogenic tissue transfer, many sterilization techniques have been used
to prevent infection through allografts which include gamma irradiation, ethylene oxide gas,
thermal treatment with moist heat, beta-propiolactone, chemical processing, and antibiotic
soaks [4]. Among these, gamma irradiation oers a clear advantage in terms of safety com-
pared with other sterilization techniques.
2.3. Xenografts
Xenograft is a term used to describe a surgical graft of tissue from one species to an unlike
species such as coral, bovine, and porcine and are used as calcied matrices generally. The
processing of xenografts is reported to remove organic components such as cells and protein-
aceous materials, leaving an inert absorbable bone scaold, which assists in revascularization,
osteoblast migration, and new bone formation [2, 6].
The use of xenografts has been demonstrated to be eective for increasing bone height and
bone volume especially in sinus augmentation procedures (Figure 1). Xenogeneic bone is
available in greater supply and larger sizes, and their physical properties are comparable to
human cancellous bone [2, 6–8]. In the literature, it has been suggested that the resorption of
xenografts and their replacement with new bone appears to be slow [9] and consideration
must be given to the risk of cross contamination with bovine spongiform encephalopathy or
porcine endogenous retroviruses [10].
2.4. Autografts
In the reconstruction of bony defects of the jaws, autogenous cancellous bone grafts are stated
to be the most eective bone graft material considering their osteoinductive eects and pre-
dictable long-term results. Autogenous bone contains all of the elements necessary to promote
vital bone formation, including mineral, collagen matrix, growth factors, and particularity
vital cells [2]. Following the transplantation, few mature osteoblasts survive the procedure,
but adequate numbers of precursor cells which have the osteogenic potential remain [2, 11].
Considering the bone volume needed, the donor sites for the reconstruction of the defects of
the jaws are anterior or posterior iliac crest, mandibular ramus, mandibular symphysis, tibia,
and parietal bone.
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Limitations of the use of autogenous bone graft harvesting dier from the selected donor
site, however, are mostly related to the so-called “donor site morbidity.” The complications
related to bone graft harvesting are [12]:
• increased operative time,
limited availability and signicant morbidity related to the intraoperative blood loss,
• wound complications,
Figure 1. Augmentation of the atrophic posterior maxilla (a) The insucent bone volume at the right posterior maxilla.
(b) Intraoral clinical view (c) Sinus bone grafting with xenograft (Bioss®, Geistlich Germany). Implants were inserted
simultaneously with sinus membrane elevation. (d) Panoramic radiograph after 1 year.
Mesenchymal Stem Cells - Isolation, Characterization and Applications188
Mesenchymal Stem Cells for Optimizing Bone Volume at the Dental Implant Recipient Site
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They have demonstrated that periosteal cells are the best choice for enhancing bone for-
mation in tissue engineering of bone regeneration. In addition, recent studies showed a
lower osteogenic dierentiation potential of adipose tissue–derived stromal cells (ASCs)
compared to bone marrow–derived mesenchymal stromal cells. According to Açil et al.
[24], a careful reconsideration of the use of ASCs in bone tissue engineering application
should be given.
3.1. Characterization of mesenchymal stem cells
Surface antigen expression, which allows for a rapid identication of a cell population, has
been extensively used in experimental studies focusing on the identication of MSCs. For
analysis of surface antigen expression, ow cytometry analysis and immunocytochemistry are
ecient methods that reveal the marker prole of individual cells. In addition, uorescence-
activated cell sorting (FACS) is a valuable protocol for sorting isolation of MSCs. (Figure 3)
All techniques described above rely on both positive and negative selection by cell antigen
surface markers, as well as physical properties of cells such as forward and side scaer char-
acteristics [25].
According to the Mesenchymal and Tissue Stem Cell Commiee of the International Society
for Cellular Therapy [26], minimal criteria to dene human MSCs are as follows:
• MSC must be plastic-adherent when maintained in standard culture conditions.
Figure 2. Bone marrow aspiration.
Mesenchymal Stem Cells - Isolation, Characterization and Applications190
MSC must express CD105, CD73, and CD90 and lack expression of CD45, CD34, CD14 or
CD11b, CD79a, or CD19 and human leukocyte antigen-D-related (HLA-DR) surface molecules.
MSC must dierentiate to osteoblasts, adipocytes, and chondroblasts in vitro.
Since the rst description of the above mentioned criteria by Mesenchymal and Tissue Stem
Cell Commiee of the International Society for Cellular Therapy in 2006, many studies have
investigated the surface antigen expression of human MSCs in order to increase the con-
dence in their identication and verication. Lee et al. [27] have demonstrated that CD14,
CD31, CD34, CD45, CD49d, CD49f, CD51, CD54, CD71, CD106, CD133, major histocompat-
ibility complex (MHC II), cytokeratin, and desmin were absent from human MSCs, whereas
CD13, CD29, CD44, CD59, CD73, CD90, CD105, CD166, MHC I,a-SMA, and vimentin were
present on human MSCs. For human bone marrow stromal cells, common targets of nega-
tive antigene expression include CD2, CD3, CD11b/Integrin alpha M, CD14, CD15/Lewis X,
CD16/Fcgamma RIII, CD19, CD38, CD56/NCAM-1, CD66b/CEACAM-8, CD123/IL-3 R alpha,
and CD235a/Glycophorin.
For the positive selection of MSCs, CD271/NGF R, CD105/Endoglin, STRO-1, ganglioside
GD2, and SUSD2 are relatively newly identied surface markers. In addition, STRO-1, CD271,
CD200, ganglioside GD2, and frizzled-9 tissue non-specic alkaline phosphatase (TNAP) are
suggested to be the latest markers used to verify MSC Identity [28, 29]. Identication of both
positive and negative novel antigen surface markers would lead to modications in the future.
Figure 3. Morphology of MSCs obtained from bone marrow transfected with uorescent protein. (Scale 100 μm).
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4. Current concepts in mesenchymal stem cell harvest
4.1. Periodontal ligament
Shi et al. [14] have showed that periodontal ligament regeneration involves the recruitment
of progenitor cells or stem cells, dierentiating into either broblasts, cementoblasts, or
osteoblasts, securing the teeth in the sockets between the cementum and adjacent alveolar
bone. Seo et al. [30] have isolated stem cells from periodontal ligament for the rst time
and gave us new strategy to reconstruction of periodontium. According to Seo et al. [30],
periodontal ligament stem cells (PDLSCs) share similar characteristics with other adult
stem cells, including the ability to self-renew and multi-lineage dierentiation potential.
All these results suggested that PDLSCs might belong to a unique population of postnatal
mesenchymal cells.
A literature survey could reveal that third molar teeth were mostly used for PDLSC isola-
tion. Briey [31], impacted third molars were surgically extracted, and periodontal dental
ligament was gently scraped from the middle root surface. Coronal and apical portions of
the ligament were not used in order to avoid contamination by gingival and pulpal cells.
Periodontal dental ligament tissues were then minced then digested in a solution contain-
ing 3 mg/ml collagenase type I and 2.5 mg/ml dispase II for 1 h at 37°C. After digestion,
tissue was seeded into culture asks with alpha-modication of Eagle’s Medium supple-
mented with 10% fetal bovine serum, 2-mM Glutamine, 100-U/mL penicillin and 100-μg/mL:
streptomycin solution at 37°C in 5% CO2 in a humidied atmosphere. After single cells were
aached on the plastic boom of the ask, non-adherent cells were removed by changing the
medium [31].
Hakki et al. [32] have suggested that BMP-2, -6, and -7 are potent regulators of periodon-
tal ligament stem cell gene expression and bio mineralization. BMPs with periodontal liga-
ment stem cell isolated from periodontal ligament tissues provide a promising strategy for
bone tissue engineering. According to a recent study performed by Açil et al. [51], BMP-7
triggers periodontal dental ligament cells to dierentiate toward an osteoblast/cementoblast
phenotype.
4.2. Adipose tissue
According to Açil et al. [24], ASCs could be easily isolated by using the modied technique that
has been previously described by Zuk et al. [33]. Briey description of the technique is; the adi-
pose tissue, which could be obtained from liposuction procedures or from the subcutaneous tis-
sue at the surgical access to the iliac crest during reconstructive maxillofacial surgical procedures.
Recent studies indicated a lower osteogenic dierentiation potential of ASCs compared to
bone marrow–derived mesenchymal stromal cells. As we have mentioned before, Açil et al.
[24] have evaluated the eects of potent combinations of highly osteogenic BMPs in order to
enhance the osteogenic dierentiation potential of ASCs and indicated a restricted osteogenic
dierentiation potential of ASCs and suggest careful reconsideration of their use in bone tis-
sue engineering applications.
Mesenchymal Stem Cells - Isolation, Characterization and Applications192
4.3. Bone marrow
The superior iliac crest is usually preferred as a donor site due to its ease in access and trabecu-
lar structure. As described by Hernigou et al. [34] and later by Shapiro et al. [35], briey, appro-
priate local anesthesia of the skin and subcutaneous soft tissues should be administered. Then,
a 1-cm stab incision was performed over iliac crest. An 11-gauge, 11-cm Jamshidi needle was
used to aspirate the bone marrow. Eort was taken to use a parallel approach, with the needle
directed parallel to the iliac wing between the inner and outer tables, and the needle was subse-
quently withdrawn and repositioned [34, 35] (Figure 4). The marrow aspirates was then passed
through a sterile lter into a separate compartment to remove particulate maer. The material
was transferred for centrifugation resulting marrow cell concentration [34, 35].
Recent literatures have showed the potential benets of using a cocktail of mononuclear cells
without expanding them in vitro before reimplantation [36] (Figure 5). Therefore, there are
also various systems developed for harvesting of MSCs from bone marrow. One of these is the
bone marrow–derived MNCs isolation by synthetic poylsaccharid (FICOLL), technique, which
is currently accepted as the gold standard [36, 37]. The FICOLL method might present a useful
Figure 4. Bone marrow aspiration from the superior iliac crest.
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technique for hospitals; however, the system is a time-consuming one, and a good manufac-
turing practice (GMP) laboratory is required. To ensure the clinical use in operating facilities
without GMP possibilities, closed systems such as closed bone marrow aspirate concentrate
(SmartPReP2 Bone Marrow Aspirate Concentrate System; BMAC; Harvest Technologies
GmbH) system were developed [38]. Saurbier et al. [36] have compared new bone formation in
maxillary sinus augmentation procedures using biomaterial associated with MSCs separated
by FICOLL and BMAC and observed a higher proportion of hard tissue in the BMAC group.
Marx et al. [39] have compared the histologic parameters and outcomes of two types of grafts in
large vertical maxillary defects: a composite graft of recombinant human bone morphogenetic pro-
tein-2/acellular collagen sponge (rhBMP-2/ACS), crushed cancellous freeze-dried allogeneic bone
(CCFDAB), and platelet-rich plasma (PRP) and size-matched 100% autogenous grafts in 20 patients.
According to their ndings, the composite graft of rhBMP-2/ACS-CCFDAB-PRP regenerates bone
in large vertical ridge augmentations as predictably as 100% autogenous graft with less morbidity,
equal cost, and more viable new bone formation without residual nonviable bone particles but with
more edema which might be aributed to the incisional release of the periosteum intraoperatively.
4.4. Peripheral blood
According to the material and methods of the experimental study performed by Sato et al. [40],
peripheral blood could be obtained by jugular vein puncture, collected into syringes containing
0.5-ml sodium heparin and should be transported at 4°C to the laboratory within 3 h. To isolate
peripheral blood-derived mononuclear cells, undiluted blood layered onto 12-ml Lympholyte
Figure 5. Diagrammatic illustration of the steps in osteoblast cell culture. Bone particles were obtained, the soft tissues
were removed, and washed with PBS. The bone particles were minced and placed in culture asks. After 3–4 weeks in
incubation, cells have reached conuence.
Mesenchymal Stem Cells - Isolation, Characterization and Applications194
in a 50-ml tube and centrifuged at 300 g for 40 min without braking [59]. The mononuclear cells
were collected and washed twice with phosphate buered saline (PBS) by centrifuging at 300 g
for 5 min followed by an additional wash with PBS. After that, cells were re-suspended in culture
medium which consists of Dulbecco’s modied Eagle’s medium with 5% separated autologous
plasma, 10% fetal bovine serum and 10-μl/ml 100-units/ml Penicillin/Streptomycin solution.
Subsequently, cells obtained from each 12 ml of blood were seeded onto a 100-mm2 tissue cul-
ture dishes and incubated in a humidied atmosphere at 37°C with 5% CO2. Nonadherent cells
were removed by washing the mononuclear cells twice with PBS after 72 h of incubation. After
2 weeks, colonies of adherent broblast-like cells could be noticed. When the colony reached the
approximate size of 5 cm2, cells are detached and seeded in a new ask. The MSCs maintained
in growth medium until ~70% conuence. The cells were then treated with 0.05% EDTA solu-
tion and could be cultured for subsequent passage in 100 mm2 dishes at 7500 cells/cm2 in base
medium. This procedure was repeated as many times as possible.
Kassis et al. [41] evaluated the ability of brin microbeads (FMB) to separate human MSC
from dierent sources other than bone marrow, with special emphasis on granulocyte colony-
stimulating factor (G-CSF)–mobilized peripheral blood of healthy individuals. According to
their material and methods, brin microbeads that bind matrix-dependent cells were pro-
duced from concentrated brinogen by a stirred heated oil emulsion technique and used to
isolate MSC from the mononuclear fraction of mobilized peripheral blood of adult healthy
human donors treated with G-CSF. Based on their results, FMB may have special advantage
in isolating MSC from mobilized peripheral blood.
The isolation of MSCs from peripheral blood is a relatively new method with the main advan-
tage of the ease in access, and further studies are needed to clarify the most appropriate tech-
nique. In addition, the introduction of platelet aggregates in oral and maxillofacial surgery
has changed the approach toward extensive reconstruction of resorbed maxillae (Figure 6)
and mandibles for implant reconstruction [42].
Figure 6. Second generation platelet aggregate (platelet rich brin) application in augmentation of the posterior maxilla.
(a) Elevation of the membrane and preparation of PRF combined with Xenograft (Bioss®, Geistlich Germany) (b) Platelet
rich brin (PRF) and Xenograft in situ. Preparation of the PRF membrane. (c) Placement of the membrane.
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A possible role of platelet aggregates in local regulation of fracture healing and bone regener-
ation was aributed to the synergic eect of growth factors such as isomers of platelet-derived
growth factor, transforming growth factor A1 and A2, insulin like growth factor > and A, and
vascular endothelial growth factor. From this point of view, platelet aggregates could help
in the dierentiation and chemotactic and mitogenic stimulation of MSCs, which leads to an
enhancement of bone repair and regeneration. Moreover, Marx [43] have recently conrmed
that platelet rich plasma (PRF) contains an amount of 250×103 −400×103 per mL, which are
positive for CD 44, CD 90, CD 105, and CD 34.
5. Mesenchymal stem cells in the reconstruction of the osseous defects of
the jaws
Tissue-engineering therapy is a recent treatment modality in dental eld to rehabilitate quan-
titative and qualitative properties of both soft and hard tissues with the use of cells with
regenerative potential signaling molecules such as growth factors and a biocompatible matrix
serving as a scaold [44, 45].
During the past 2 decades, various experimental studies focusing on the osteogenic prop-
erties of MSCs have been performed. In 2001, Cooper et al. [46] have studied the relation-
ship between bone sialoprotein (BSP) expression and osteocalcin expression with subsequent
osteogenesis occurring in MSC-based implants and suggested that culture-expanded, cryo-
preserved human MSCs have osteogenic potential and demonstrated that implanted cell gene
expression can reveal the early onset of bone formation.
In 2003, De Kok et al. [47] have evaluated MSC-based alveolar bone regeneration in a canine
alveolar saddle defect model and observed that equivalent amounts of new bone were formed
within the pores of the matrices loaded with autologous MSCs or MSCs from an unrelated
donor, conrming the hypothesis that MSCs have the capacity to regenerate bone within
craniofacial defects In addition, they have also stated that neither autologous nor allogeneic
MSCs induced a systemic response by the host. Gutwald et al. [48] compared the osteogenic
potential of mononuclear cells harvested from the iliac crest combined with bovine bone min-
eral (BBM) with that of autogenous cancellous bone alone and studied bilateral augmenta-
tions of the sinus oor in 6 adult sheep and reported that MSCs, in combination with BBM as
the biomaterial, have the potential to form bone.
In the literature, there are also numerous studies focusing on the stimulating eects of various
growth factors, most notably BMPs, on the osteogenic dierentiation of MSCs [49, 50]. Açil et
al. [51] have compared the most potent growth factors in regard to their osteoinductive poten-
tial and stated that the combined addition of BMP-2, BMP-6, and BMP-9 to the osteoinductive
culture medium containing dexamethasone, β-glycerophosphate, and ascorbate-2-phosphate
produces more potent osteoblast dierentiation of human MSCs in vitro.
Following various experimental studies, the number of the clinical prospective studies has
also increased steadily, and good cases of translational research from basic research to clinical
Mesenchymal Stem Cells - Isolation, Characterization and Applications196
application have arisen. In a groundbreaking study, Wiltfang et al. [52] have reconstructed
a mandibular discontinuity defect after ablative surgery using the gastrocolic omentum as
a bioreactor for heterotopic ossication via a titanium mesh cage lled with bone mineral
blocks, inltrated with 12 mg of recombinant human BMP2, and enriched with bone marrow
aspirate. The scaold was implanted into the gastrocolic omentum, and 3 months later, a
free ap was harvested to reconstruct the mandibular defect. In vivo single-photon-emission
computed tomography/computed tomography revealed bone remodeling and mineralization
inside the mandibular transplant during prefabrication. They have reported that the quality
of life of the patient signicantly increased with acquisition of the ability to masticate and the
improvement in pronunciation and aesthetics.
It is well known that MSCs can be directed to dierentiate into an osteoblastic lineage in
the presence of growth factors. Furthermore, platelet-rich plasma (PRP), which can be easily
isolated from whole blood, was often used for bone regeneration, wound healing, and bone
defect repair [53]. Marx [43] have stated that PRP contains an amount of 250×103 −400×103 per
ml which are positive for CD 44, CD 90, CD 105, and CD 34.
Yamada et al. [54] investigated as basic research tissue-engineered bone regeneration using
MSCs and PRP in a dog mandible model and conrmed the correlation between osseointegra-
tion in dental implants and the injectable bone. After that, same authors applied this injectable
tissue-engineered bone to onlay plasty in the posterior maxilla or mandible in three human
patient with simultaneous implant placement and reported stable and predictable results in
terms of implant success [55]. In 2005, Ueda et al. [56] have used MSCs in a clinical study under-
taken to evaluate the use of tissue-engineered bone, MSCs, platelet-rich plasma, and beta-trical-
cium phosphate as grafting materials for maxillary sinus oor augmentation and proclaimed
that tissue-engineered bone provided stable and predictable results in terms of implant success.
In order to increase the amount of available bone where dental implants must be placed, Filho
Cerruti et al. [57] evaluated PRP and mononuclear cells (MNCs) from bone marrow aspirate
and bone scaold in 32 patients and have concluded that the process of healing observed in the
patients was due to the presence of mesenchymal stem cell in MNC fraction in the bone grafts.
Schmelzeisen et al. [58] reported a simplied method of using to regenerate hard tissue and sug-
gested that bone marrow aspirate concentrate combined with a suitable biomaterial can form
sucient bone within 3 months for further implants to be inserted and at the same time mini-
mize morbidity at the donor site. Similarly, Ricket et al. [59] have assessed whether dierences
occur in bone formation after maxillary sinus oor elevation surgery with bovine bone mineral
mixed with autogenous bone or autogenous stem cells and stated that MSCs seeded on bovine
bone mineral particles can induce the formation of a sucient volume of new bone to enable
the reliable placement of implants within a time frame comparable with that of applying either
solely autogenous bone or a mixture of autogenous bone and bovine bone mineral particles.
Not only the defects at implant recipient sites, peri-implantar bone loss has also become a
point of interest for some researchers, and eorts have been made over the last few decades
to produce reliable and predictable methods to stimulate bone regeneration in bone defects
resulting from peri-implant diseases [60]. Ribeiro et al. [61] have investigated the eect of bone
marrow–derived cells associated with guided bone regeneration in the treatment of dehiscence
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bone defects around dental implants and suggested that bone marrow–derived cells provided
promising results for peri-implantar bone regeneration, although the combined approach
seems to be relevant, especially to bone formation out of the implant threads. Similarly, Kim et
al. [62] evaluated the potential of periodontal ligament stem cells and bone marrow stem cells
on alveolar bone regeneration in a canine peri-implant defect model and demonstrated the
feasibility of using stem cell–mediated bone regeneration to treat peri-implant defects.
6. Conclusion
A growing number of studies indicate that stem applications are feasible protocols with clin-
ically successful results in restoration of the bone architecture of the maxillofacial region.
Composite grafts of MSCs, BMP, PRP, and bone graft combinations are able to achieve clinical
results equivalent to autogenous grafts in large vertical ridge augmentations without donor
bone harvesting.
Continued and extended experimental studies are needed to exactly determine the isolation,
characterization, and dierentiation properties of MSCs. In addition, development of chair-
side protocols would be benecial in order to adapt MSC applications to the daily dental
practice.
Abbreviations
ASC Adipose tissue-derived stromal cells
BMAC Bone marrow aspirate concentrate
BMP Bone morphogenetic protein
CCFDAB Crushed cancellous freeze-dried allogeneic bone
CD Cluster of dierentiation
CEACAM Carcinoembryonic antigen-related cell adhesion molecule
FACS Fluorescence-activated cell sorting
FMB Fibrin microbeads
GMP Good manufacturing practice
HIV Human immunodeciency virus
HLA-DR Human leukocyte antigen-D-related
MHC Major histocompatibility complex
MSC Mesenchymal stem cell
NCAM Neural cell adhesion molecule
PBS Phosphate buered solution
Mesenchymal Stem Cells - Isolation, Characterization and Applications198
PDLSC Periodontal ligament stem cell
PRF Platelet rich plasma
PRP Platelet-rich brin
rhBMP Recombinant human bone morphogenetic protein
TNAP Tissue non-specic Alkaline Phosphatase
Author details
Mustafa Ayna1,3*, Aydin Gülses2, Jörg Wiltfang3,4 and Yahya Açil3,4
*Address all correspondence to: praxis@dr-ayna.de
1 Danube University Krems, Krems, Austria
2 Ankara 2nd Division Public Hospital Association, Ankara, Turkey
3 Center for Oral Surgery and Implantology, Duisburg, Germany
4 Department of Oral and Maxillofacial Surgery, Christian Albrechts University, Kiel,
Germany
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The aim of this study was to isolate mesenchymal stem cells (MSCs) from feline peripheral blood (fPB-MSCs) and to characterise the cells' in vitro properties. The mononuclear cell fractions were isolated from venous blood of cats by density gradient centrifugation and cultured on plastic dishes under various culture conditions to isolate MSCs. When these cells were cultured with 5% autologous plasma (AP) and 10% foetal bovine serum (FBS), adherent spindle shaped fibroblast-like cells (fPB-MSCs) were obtained from 15/22 (68%) cats. These cells were isolated only from medium containing both AP and FBS. The morphology of these MSCs was similar to those isolated from other species and from other feline tissues. fPB-MSCs expanded steadily up to 5–6 passages, but had increased population doubling time during passaging and almost all cells stopped proliferation at passages 7–9. These cells expressed CD44 and CD90, and were mostly negative for major histocompatibility class II and CD4. The cells could be induced to differentiate into adipogenic, osteogenic and chondrogenic cell lineages. These findings indicate that fPB-MSCs can be generated but appear to require specific culture conditions.
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Background: Bone marrow aspirate concentrate (BMAC) is increasingly used as a regenerative therapy for musculoskeletal pathological conditions despite limited evidence-based support. Hypothesis: BMAC will prove feasible, safe, and efficacious for the treatment of pain due to mild to moderate degenerative joint disease of the knee. Study design: Randomized controlled trial; Level of evidence, 2. Methods: In this prospective, single-blind, placebo-controlled trial, 25 patients with bilateral knee pain from bilateral osteoarthritis were randomized to receive BMAC into one knee and saline placebo into the other. Fifty-two milliliters of bone marrow was aspirated from the iliac crests and concentrated in an automated centrifuge. The resulting BMAC was combined with platelet-poor plasma for an injection into the arthritic knee and was compared with a saline injection into the contralateral knee, thereby utilizing each patient as his or her own control. Safety outcomes, pain relief, and function as measured by Osteoarthritis Research Society International (OARSI) measures and the visual analog scale (VAS) score were tracked initially at 1 week, 3 months, and 6 months after the procedure. Results: There were no serious adverse events from the BMAC procedure. OARSI Intermittent and Constant Osteoarthritis Pain and VAS pain scores in both knees decreased significantly from baseline at 1 week, 3 months, and 6 months ( P ≤ .019 for all). Pain relief, although dramatic, did not differ significantly between treated knees ( P > .09 for all). Conclusion: Early results show that BMAC is safe to use and is a reliable and viable cellular product. Study patients experienced a similar relief of pain in both BMAC- and saline-treated arthritic knees. Further study is required to determine the mechanisms of action, duration of efficacy, optimal frequency of treatments, and regenerative potential. Registration: ClinicalTrials.gov record 12-004459.