The Progress of Stem Cell Technology for Skeletal Regeneration.

Skeletal disorders, such as osteoarthritis and bone fractures, are among the major conditions that can compromise the quality of daily life of elderly individuals. To treat them, regenerative therapies using skeletal cells have been an attractive choice for patients with unmet clinical needs. Currently, there are two major strategies to prepare the cell sources. The first is to use induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs), which can recapitulate the skeletal developmental process and differentiate into various skeletal cells. Skeletal tissues are derived from three distinct origins: the neural crest, paraxial mesoderm, and lateral plate mesoderm. Thus, various protocols have been proposed to recapitulate the sequential process of skeletal development. The second strategy is to extract stem cells from skeletal tissues. In addition to mesenchymal stem/stromal cells (MSCs), multiple cell types have been identified as alternative cell sources. These cells have distinct multipotent properties allowing them to differentiate into skeletal cells and various potential applications for skeletal regeneration. In this review, we summarize state-of-the-art research in stem cell differentiation based on the understanding of embryogenic skeletal development and stem cells existing in skeletal tissues. We then discuss the potential applications of these cell types for regenerative medicine.

Did Osteoblastic Cell Therapy Improve the Prognosis of Pre-fracture Osteonecrosis of the Femoral Head? A Randomized, Controlled Trial.

BACKGROUND: In patients with nontraumatic osteonecrosis of the femoral head (ONFH), implantation of bone marrow aspirate concentrate (BMAC) could delay the progression of osteonecrosis and improve symptoms in pre-fracture ONFH. However, the BMAC content, especially in osteoblastic stem cells, could have an important individual variability. An autologous osteoblastic cell product could improve the effect of such cell-based therapy. QUESTIONS/PURPOSES: (1) Does autologous osteoblastic cell therapy decrease the likelihood of progression to subchondral fracture with or without early collapse corresponding to Association Research Circulation Osseous (ARCO) classification Stage III or higher, and provide a clinically important pain improvement compared with BMAC treatment alone? (2) Were patients treated with osteoblastic cell therapy less likely to undergo subsequent THA? (3) What proportion of patients in the treatment and control groups experienced adverse events after surgery? METHODS: Between 2004 and 2011, we treated 279 patients for Stage I to II hip osteonecrosis (ON) with surgery. During that time, our general indications for surgery in this setting included non-fracture ON lesions. To be eligible for this randomized, single-blind trial, patients needed to have an ONFH Stage I or II; we excluded those with traumatic ONFH, hemoglobinopathies and positive serology for hepatitis B, C or HIV. Of those treated surgically for this diagnosis during the study period, 24% (67) agreed to participate in this randomized trial. Hips with pre-fracture ONFH were randomly treated with a core decompression procedure associated with either implantation of a BMAC (BMAC group; n = 26) or osteoblastic cell (osteoblastic cell group; n = 30). The groups were not different in terms of clinical and imaging characteristics. The primary study outcome was treatment response, defined as the absence of progression to subchondral fracture stage (ARCO stage III or higher) plus a clinically important pain improvement defined as 1 cm on a 10-cm VAS. The secondary endpoint of interest was the frequency in each group of subsequent THA and the frequency of adverse events. The follow-up duration was 36 months. We used an as-treated analysis (rather than intention-to-treat) for our efficacy endpoint, and an intention-to-treat analysis for adverse events. Overall, 26 of 26 patients in the BMAC group and 27 of 30 in the osteoblastic cell group completed the trial. RESULTS: At 36 months, no clinically important differences were found in any study endpoint. There was no difference in the proportion of patients who had progressed to fracture (ARCO stage III or higher; 46% of the BMAC hips [12 of 26] versus 22% in the hips with osteoblastic cells [six of 27], hazard ratio, 0.47 [95% CI 0.17 to 1.31]; p = 0.15). There was no clinically important difference in VAS pain scores. No differences were found for either the WOMAC or the Lequesne indexes. With the numbers available, there was no difference in the proportion of patients in the groups who underwent THA at 36 months 15% (four of 27) with osteoblastic cells versus 35% (nine of 26) with BMAC; p = 0.09 With the numbers available, we found no differences between the treatment and control groups in terms of the frequencies of major adverse events. CONCLUSIONS: We found no benefit to osteoblastic cells over BMAC in patients with pre-collapse ONFH; side effects were uncommon and generally mild in both groups. This study could be used as pilot data to help determine sample sizes for larger (presumably multicenter) randomized controlled trials. However, this novel treatment cannot be recommended in routine practice until future, larger studies demonstrate efficacy. LEVEL OF EVIDENCE: Level II, therapeutic study.

Interplay between microRNAs and Wnt, transforming growth factor-ß, and bone morphogenic protein signaling pathways promote osteoblastic differentiation of mesenchymal stem cells.

Osteoblasts are terminally differentiated cells with mesenchymal origins, known to possess pivotal roles in sustaining bone microstructure and homeostasis. These cells are implicated in the pathophysiology of various bone disorders, especially osteoporosis. Over the last few decades, strategies to impede bone resorption, principally by bisphosphonates, have been mainstay of treatment of osteoporosis; however, in recent years more attention has been drawn on bone-forming approaches for managing osteoporosis. MicroRNAs (miRNAs) are a broad category of noncoding short sequence RNA fragments that posttranscriptionally regulate the expression of diverse functional and structural genes in a negative manner. An accumulating body of evidence signifies that miRNAs direct mesenchymal stem cells toward osteoblast differentiation and bone formation through bone morphogenic protein, transforming growth factor-ß, and Wnt signaling pathways. MiRNAs are regarded as excellent future therapeutic candidates because of their small size and ease of delivery into the cells. Considering their novel therapeutic significance, this review discusses the main miRNAs contributing to the anabolic aspects of bone formation and illustrates their interactions with corresponding signaling pathways involved in osteoblastic differentiation.

Cytoprotective Preconditioning of Osteoblast-Like Cells with N-Acetyl-L-Cysteine for Bone Regeneration in Cell Therapy.

Oxidative stress hinders tissue regeneration in cell therapy by inducing apoptosis and dysfunction in transplanted cells. N-acetyl-L-cysteine (NAC) reinforces cellular antioxidant capabilities by increasing a major cellular endogenous antioxidant molecule, glutathione, and promotes osteogenic differentiation. This study investigates the effects of pretreatment of osteoblast-like cells with NAC on oxidative stress-induced apoptosis and dysfunction and bone regeneration in local transplants. Rat femur bone marrow-derived osteoblast-like cells preincubated for 3 h with and without 5 mM NAC were cultured in a NAC-free osteogenic differentiation medium with continuous exposure to 50 µM hydrogen peroxide to induce oxidative stress. NAC preincubation prevented disruption of intracellular redox balance and alleviated apoptosis and negative impact on osteogenic differentiation, even under oxidative stress. Autologous osteoblast-like cells with and without NAC pretreatment in a collagen sponge vehicle were implanted in critical-size defects in rat femurs. In the third week, NAC-pretreated cells yielded complete defect closure with significantly matured lamellar bone tissue in contrast with poor bone healing by cells without pretreatment. Cell-tracking analysis demonstrated direct bone deposition by transplanted cells pretreated with NAC. Pretreatment of osteoblast-like cells with NAC enhances bone regeneration in local transplantation by preventing oxidative stress-induced apoptosis and dysfunction at the transplanted site.

Effect of cell therapy with allogeneic osteoblasts on bone repair of rat calvaria defects.

BACKGROUND AIMS: Regenerative medicine strategies based on cell therapy are considered a promising approach to repair bone defects. The aims of this study were to evaluate the effect of subculturing on the osteogenic potential of osteoblasts derived from newborn rat calvaria and the effect of these osteoblasts on bone repair of rat calvaria defects. METHODS: Cells were obtained from 50 newborn rat calvaria, and primary osteoblasts (OB) were compared with first passage (OB-P1) in terms of osteogenic potential by assaying cell proliferation, alkaline phosphatase (ALP) activity, extracellular matrix mineralization and gene expression of the osteoblastic markers RUNX2, ALP, osteocalcin and bone sialoprotein. Then, 5-mm calvaria defects were created in 24 Wistar rats, and after 2 weeks, they were locally injected with 50 µL of phosphate-buffered saline containing either 5 × 106 osteoblasts (OB-P1, n = 12) or no cells (control, n = 12). Four weeks post-injection, the bone formation was evaluated by micro-computed tomography and histological analyses. Data were compared by analysis of variance, followed by the Student-Newman-Keuls's test or Student's t-test (P ≤ 0.05). RESULTS: OB-P1 showed high proliferation and ALP activity, and despite the reduced gene expression of osteoblastic markers and extracellular matrix mineralization compared with OB, they displayed osteogenic potential, being a good choice for injection into calvaria defects. The micro-tomographic and histological data showed that defects treated with OB-P1 presented higher bone formation compared with control defects. DISCUSSION: Our results indicate that cells derived from newborn rat calvaria retain osteoblastic characteristics after subculturing and that these osteoblasts stimulate bone repair in a rat calvaria defect model.

Peculiarities of Osteogenesis by Periosteal Cells after Experimental Ectopic Transplantation.

We carried out a comparative study of the features of osteogenesis from the progenitor osteogenic periosteal cells in rabbit and human. At the initial stages, high osteogenic potential of both human and rabbit periosteal cells was observed. However, at the later stages, the cell response favors resorption of the new bone tissue formed from periosteal cells in rabbits, but does not affect the bone tissue formed from human progenitor osteogenic periosteal cells. These functional characteristics of rabbit periosteal cells should be considered when planning the experiment.

Challenges in engineering large customized bone constructs.

The ability to treat large tissue defects with customized, patient-specific scaffolds is one of the most exciting applications in the tissue engineering field. While an increasing number of modestly sized tissue engineering solutions are making the transition to clinical use, successfully scaling up to large scaffolds with customized geometry is proving to be a considerable challenge. Managing often conflicting requirements of cell placement, structural integrity, and a hydrodynamic environment supportive of cell culture throughout the entire thickness of the scaffold has driven the continued development of many techniques used in the production, culturing, and characterization of these scaffolds. This review explores a range of technologies and methods relevant to the design and manufacture of large, anatomically accurate tissue-engineered scaffolds with a focus on the interaction of manufactured scaffolds with the dynamic tissue culture fluid environment. Biotechnol. Bioeng. 2017;114: 1129-1139. © 2016 Wiley Periodicals, Inc.

Prolonged Survival of Transplanted Osteoblastic Cells Does Not Directly Accelerate the Healing of Calvarial Bone Defects.

Considering the increased interest in cell-based bone regeneration, it is necessary to reveal the fate of transplanted cells and their substantive roles in bone regeneration. The aim of this study was to analyze the fate of transplanted cells and the effect of osteogenic cell transplantation on calvarial bone defect healing. An anti-apoptotic protein, heat shock protein (HSP) 27, was overexpressed in osteoblasts. Then, the treated osteoblasts were transplanted to calvarial bone defect and their fate was analyzed to evaluate the significance of transplanted cell survival. Transient overexpression of Hsp27 rescued MC3T3-E1 osteoblastic cells from H2 O2 -induced apoptosis without affecting osteoblastic differentiation in culture. Transplantation of Hsp27-overexpressing cells, encapsulated in collagen gel, showed higher proliferative activity, and fewer apoptotic cells in comparison with control cells. After 4-week of transplantation, both control cell- and Hsp27 overexpressed cell-transplanted groups showed significantly higher new bone formation in comparison with cell-free gel-transplantation group. Interestingly, the prolonged survival of transplanted osteoblastic cells by Hsp27 did not provide additional effect on bone healing. The transplanted cells in collagen gel survived for up to 4-week but did not differentiate into bone-forming osteoblasts. In conclusion, cell-containing collagen gel accelerated calvarial bone defect healing in comparison with cell-free collagen gel. However, prolonged survival of transplanted cells by Hsp27 overexpression did not provide additional effect. These results strongly indicate that cell transplantation-based bone regeneration cannot be explained only by the increment of osteogenic cells. Further studies are needed to elucidate the practical roles of transplanted cells that will potentiate successful bone regeneration. J. Cell. Physiol. 231: 1974-1982, 2016. © 2016 Wiley Periodicals, Inc.

Comparing immunocompetent and immunodeficient mice as animal models for bone tissue engineering.

OBJECTIVES: To understand the differences and similarities between immunocompetent and immunodeficient mice as ectopic transplantation animal models for bone tissue engineering. MATERIALS AND METHODS: Osteogenic cells from mouse leg bones were cultured, seeded on ß-TCP granules, and transplanted onto the backs of either immunocompetent or immunodeficient nude mice. At 1, 2, 4, and 8 weeks postoperatively, samples were harvested and evaluated by hematoxylin-eosin staining, tartrate-resistant acid phosphatase (TRAP) staining, and immunohistochemical staining and quantitative PCR. RESULTS: In immunocompetent mice, inflammatory cell infiltration was evident at 1 week postoperatively and relatively higher expression of TNF-α and IL-4 was observed. In immunodeficient mice, new bone area and the number of TRAP-positive cells were larger at 4 weeks than in immunocompetent mice. The volume of new bone area in immunodeficient mice was reduced by 8 weeks. CONCLUSIONS: Bone regeneration was feasible in immunocompetent mice. However, some differences were observed between immunocompetent and immunodeficient mice in the bone regeneration process possibly due to different cytokine expression, which should be considered when utilizing in vivo animal models.

Up-and-Coming Mandibular Reconstruction Technique With Autologous Human Bone Marrow Stem Cells and Iliac Bone Graft in Patients With Large Bony Defect.

Large bony defects followed by resection of the mandible need to be reconstructed by various surgical techniques such as the fibular flap. In this article, we report the case of mandibular reconstruction with autologous human bone marrow mesenchymal stem cells and autogenous bone graft, followed by placement of dental implants and prosthodontic treatment in a patient who has been failed to reconstruct mandibular bone defect after resection of mandible.

In vitro and in vivo characterization of porcine acellular dermal matrix for gingival augmentation procedures.

BACKGROUND AND OBJECTIVE: Recently, porcine acellular dermal matrix (PADM) has been proposed as a possible alternative to autogenous grafts in periodontal plastic surgery. The aim of the present study was to investigate the in vitro responses of four different oral cell lines cultured on a novel PADM. Furthermore, tissue reaction to PADM was evaluated histologically after subcutaneous implantation in mice. MATERIAL AND METHODS: Human gingival fibroblasts (HGF), human osteoblast-like cells, human umbilical vein endothelial cells and human oral keratinocytes (HOK) were cultured and transferred on to the PADM. A tissue culture polystyrene surface served as the control. The viability of all tested cell lines on PADM was measured by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide colorimetric assay and PrestoBlue(®) reagent. The ToxiLight(®) assay was performed to analyze the effect of PADM on adenylate kinase release. PADM was implanted into nude mice subcutaneously and subjected to histological analysis after 21 d. RESULTS: Using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide colorimetric assays, all tested cell lines cultured on PADM demonstrated a significant increase of viability compared to the control group (each p < 0.001) with the exception of HGF and HOK after 3 d (each p > 0.05). According to the PrestoBlue(®) analysis, all cell lines demonstrated a significant increase of viability compared to the control group at the particular points of measurement after 18 h (HGF p < 0.01; human osteoblast-like cells, human umbilical vein endothelial cells, HOK each p < 0.001). No significant cytotoxic effects of PADM on the tested cell lines could be observed, as assessed by changes in adenylate kinase release. Subcutaneous implantation of PADM into nude mice demonstrated good integration with surrounding tissues and significant revascularization of its collagen structure. CONCLUSION: Overall, the results suggest that PADM is a promising substitute for autogenous soft tissue grafts in periodontal surgery.

Mandibular symphysis and ramus as sources of osteoblastic cells for bone tissue engineering.

OBJECTIVES: Autografts from mandibular symphysis and ramus are often used for bone reconstruction. Based on this, we hypothesized that these sites could be useful cell sources for bone tissue engineering approaches. Thus, our study aimed at evaluating the proliferation and osteoblast phenotype development of cells derived from mandibular symphysis and ramus. MATERIALS AND METHODS: Cells were isolated from bone fragments of four patients by enzymatic digestion and cultured under osteogenic condition for up to 17 days. Cultures were assayed for cell proliferation, gene expression of key bone markers runt-related transcription factor 2 (Runx2), distal-less homeobox 5 (DLX5), SATB homeobox 2 (SATB2), Osterix (OSX), family with sequence similarity 20, member C (FAM20C), bone sialoprotein (BSP), osteopontin (OPN) and osteocalcin (OC), alkaline phosphatase (ALP) expression and activity, and extracellular matrix mineralization. Data were compared by two-way ANOVA or t-test for independent samples when appropriate. RESULTS: Cells derived from ramus displayed lower proliferative activity and higher gene expression of Runx2, DLX5, SATB2, OSX, FAM20C, BSP, OPN and OC, ALP protein expression and activity and extracellular matrix mineralization compared with symphysis-derived cells. CONCLUSION: Symphysis and ramus may be considered as cell sources for bone tissue engineering approaches but due to the higher osteogenic potential, ramus-derived cells are more appealing for constructing cell-based biomaterials.

Comparison of bone dust with other types of bone grafts for cranioplasty.

PURPOSE: Split calvarial bone graft is preferred in the reconstruction of calvarial defects. However, it is not feasible for use in some challenging cases and in children. Particulate bone graft containing viable osteoblasts could be an attractive alternative. MATERIALS AND METHODS: A total of 32 female rats were randomly separated into 4 groups. Full-thickness bone graft from rat calvaria was harvested in diameters of 8 × 8 mm. In group 1, the periosteum and skin were closed without any bone graft; bone dust particles were placed in group 2; bone fragments were placed in group 3; and full-thickness cranial bone graft was placed in group 4. After 12 weeks, all rats were killed. Degrees of resorption, foreign body reaction, and bone spicule length were assessed histologically, and an immunohistochemical study was used to show bone graft viability. RESULTS: In graft viability, osteogenesis, and osteoblastic differentiation, groups 3 and 4 were similar and superior to groups 1 and 2. No osteoblastic activity and no viable bone dust were detected in groups 1 and 2. Resorption was observed in every preparate that contains bone tissue, and foreign body reaction was prominent in small bone groups, such as in group 2. CONCLUSIONS: In the full-thickness cranial bone graft group and the bone fragment group, the preservation of bone viability was obviously superior to the bone dust group and the periosteum-only group. In conclusion, bone dust behaved like the periosteum and could not create new bone, whereas bone particles behaved like the full-thickness cranial bone graft and were capable of preserving viability.

Generation of co-culture spheroids as vascularisation units for bone tissue engineering.

Cell spheroids represent attractive building units for bone tissue engineering, because they provide a three-dimensional environment with intensive direct cell-cell contacts. Moreover, they allow for co-culture of both osteoblasts and vessel-forming cells, which may markedly increase their survival and vascularisation after transplantation. To test this hypothesis, we generated co-culture spheroids by aggregating different combinations of primary human osteoblasts (HOB), human dermal microvascular endothelial cells (HDMEC) and normal human dermal fibroblasts (NHDF) using the liquid overlay technique. Mono-culture spheroids consisting either of HOB or HDMEC served as controls. After in vitro characterisation, the different spheroids were transplanted into dorsal skinfold chambers of CD1 nu/nu mice to study in vivo their viability and vascularisation over a 2-week observation period by means of repetitive intravital fluorescence microscopy and immunohistochemistry. In vitro, co-culture spheroids containing HDMEC rapidly formed dense tubular vessel-like networks within 72 h and exhibited a significantly decreased rate of apoptotic cell death when compared to mono-culture HDMEC spheroids. After transplantation, these networks interconnected to the host microvasculature by external inosculation. Of interest, this process was most pronounced in HOB-HDMEC spheroids and could not further be improved by the addition of NHDF. Accordingly, HOB-HDMEC spheroids were larger when compared to the other spheroid types. These findings indicate that HOB-HDMEC spheroids exhibit excellent properties to preserve viability and to promote proliferation and vascularisation. Therefore, they may be used as functional vascularisation units in bone tissue engineering for the seeding of scaffolds or for the vitalisation of non-healing large bone defects.

Treatment of osteonecrosis in the knee joint of a rabbit using autologous cultured osteoblasts.

BACKGROUND: [corrected] To develop a successful treatment modality for osteonecrosis, an appropriate animal model is essential. We have proposed a new osteonecrosis model that shows the total amount of necrosis and in which we observed new bone formation after transplanting autologous cultured osteoblasts. MATERIALS AND METHODS: The femoral condyles of the right knees of New Zealand white rabbits were exposed after dissecting the ligaments surrounding the distal femur. After which, the metaphyseal-diaphyseal junction was cut using a saw, and the entire femoral condyle was isolated. After three liquid nitrogen treatments, the isolated femoral condyle was internally fixated to the femoral shaft using two or three Kirschner wires. Bone marrow isolated from the iliac crest was cultivated to differentiate it into osteoblasts, and the cultured cells were then injected into the necrotic bone. RESULTS: Viable osteocytes with well-stained nuclei were not present in the necrotic areas at any stage of the development of the osteonecrosis model within 24 wk after osteonecrosis induction. However, new bone formation with osteocytes and blood vessels was observed in the necrotic bone 12 wk after transplanting the autologous cultured osteoblasts. CONCLUSIONS: The distal femoral condyle of the rabbit is an appropriate model for demonstrating osteonecrosis and treatment evaluation owing to its easy reproducibility and treatment interpretation. Therefore, autologous cultured osteoblast treatment would seem to be a potentially successful treatment modality for osteonecrosis.

Dedifferentiated follicular granulosa cells derived from pig ovary can transdifferentiate into osteoblasts.

Transdifferentiation is the conversion of cells from one differentiated cell type into another. How functionally differentiated cells already committed to a specific cell lineage can transdifferentiate into other cell types is a key question in cell biology and regenerative medicine. In the present study we show that porcine ovarian follicular GCs (granulosa cells) can transdifferentiate into osteoblasts in vitro and in vivo. Pure GCs isolated and cultured in Dulbecco's modified Eagle's medium supplemented with 20% FBS (fetal bovine serum) proliferated and dedifferentiated into fibroblast-like cells. We referred to these cells as DFOG (dedifferentiated follicular granulosa) cells. Microarray analysis showed that DFOG cells lost expression of GC-specific marker genes, but gained the expression of osteogenic marker genes during dedifferentiation. After osteogenic induction, DFOG cells underwent terminal osteoblast differentiation and matrix mineralization in vitro. Furthermore, when DFOG cells were transplanted subcutaneously into SCID mice, these cells formed ectopic osteoid tissue. These results indicate that DFOG cells derived from GCs can differentiate into osteoblasts in vitro and in vivo. We suggest that GCs provide a useful model for studying the mechanisms of transdifferentiation into other cell lineages in functionally differentiated cells.

(111)Indium-oxine labelling for evaluating the homing process of autologous osteoblasts implanted percutaneously in atrophic nonunion fractures.

PURPOSE: The aim of the study was to control the in vivo localisation of implanted cells in cell-based therapies. Labelling cells with (111)indium-oxine is one of the most interesting methods proposed. We evaluated this method in the setting of autologous osteoblast implantation in nonunion fractures. METHODS: An in vitro study of osteoblasts was conducted after (111)indium-oxine labelling. Radioactivity retention and viability, proliferation and the ability to produce alkaline phosphatase were evaluated in a seven-day culture. In vivo labelling of implanted osteoblastic cells was conducted during a therapeutic trial of atrophic nonunion fractures, with the leakage outside the nonunion site and local uptake evolution at four, 24 and 48 hour being studied. RESULTS: The mean labelling efficiency for osteoprogenitors was 78.8 ± 4.6 %. The intracellular retention was 89.4 ± 2.1 % at three hours and 67.3 ± 4.7 % at 18 hours. The viability assessed at three hours was 93.7 ± 0.6 %. After seven days of culture, morphology and alkaline phosphatase staining were similar for both labelled and unlabelled control cells, although the proliferation rate was decreased in the labelled cells. Some local intraosseous leakage was observed in four of 17 cases. All patients showed uptake at the injection site, with four having no other uptake. Four patients showed additional uptake in the bladder, liver and spleen, while 11 patients had additional uptake in the lungs in addition to the bladder, liver and spleen. The activity ratios (injection site/body) were 48 ± 28 % at four hours, 40 ± 25 % at 24 hours and 35 ± 25 % at 48 hours. After correcting for decay, the activity within the injection site was 82 ± 15 % at 24 hours and 69 ± 11 % at 48 hours compared with the activity measured at four hours. No relationship was found between uptake and radiological bone repair. CONCLUSIONS: The (111)indium-oxine labelling appears to be a good method for monitoring the behaviour of the osteoblastic cells after their implantation in atrophic nonunion fractures.

The effect of the microenvironment created by a titanium mesh cage on subcutaneous experimental bone formation and inhibition of absorption.

We attempted to form ectopic bone under the skin of rats without adding any extrinsic bone-inducing growth factors or cytokines using bone marrow stromal cells (BMSCs), a collagen scaffold and a titanium mesh cage. We set up a space made up of a cage inserted into the subcutaneous region of rats' backs, where we could eliminate the possible influence of residual bone tissue on bone induction. We filled this space with a collagen matrix containing BMSCs. At week 8 and month 6 after implantation, the specimens were removed and observed histologically, histochemically and enzyme histochemically. As a result, bone tissue was identified in each case within the titanium cages, even though we had not used bone-inducing chemical substances. Bone generation was not found in test cases without a cage. Enhanced green fluorescence protein (EGFP) labeling of the implanted BMSCs clearly showed that these cells differentiated into osteoblasts and subsequently into osteocytes in the formed bone tissue. Host cells without EGFP labeling were also confirmed to be involved in bone formation. Six months after transplantation, the implanted cells were still present in the generated bone, and no significant resorption of the generated bone was observed. These results indicate that the physically stable spatial microenvironment created by the cage in vivo plays an important role in bone formation and inhibition of its resorption, which we refer to as the 'cage effect'.

Effects of mesenchymal stem cells in critical size bone defect.

BACKGROUND AND OBJECTIVES: The aim of this study was to compare culture-expanded, bone marrow-derived mesenchymal stem cells (MSCs) and platelet-rich plasma (PRP) loaded to biphasic calcium phosphate (BCP) bone ceramic in the repair of rat calvarial bone. MATERIALS AND METHODS: Critical-size (7 mm dia.) calvarial defects were prepared in the frontal-parietal bones of 90 adult female Sprague-Dawley rats. Rats were randomly divided into 5 groups, according to defect filling, as follows: Group I (n = 21), BCP; Group II (n = 21), BCP+PRP; Group III (n = 21), BCP+MSC; Group IV (n = 21), BCP+PRP+MSC; Group V (n = 6) (control), no treatment. Animals were sacrificed at 2, 8 and 12 weeks postsurgery and bone regeneration was evaluated both histologically and immunohistochemically. RESULTS: Statistically significant differences were observed in bone osteoblastic activity in calvarial defects among the groups (p < 0.05). PRP and MSC used in combination with BCP as a defect filling resulted in greater osteoblastic bone formation activity when compared to the use of BCP alone. CONCLUSIONS: The combination of mesenchymal stem cells, platelet rich plasma and synthetic bone substitute was found to be more effective in inducing new bone formation (osteogenesis) than the use of platelet rich plasma combined with synthetic bone substitute and the use of synthetic bone substitute alone.

Stem cell therapy for bone regeneration: present and future strategies.

Alveolar bone atrophy may occur due to trauma, malignancy and periodontal disease. Restoring the lost bone is crucial for the rehabilitation of the patient's functioning, phonetics and aesthetics. Currently the methods available for vertical bone augmentation prior to dental implant placement are rather limited. This paper describes present and future concepts of utilizing mesenchymal stem cells (MSC) as well as endothelial progenitor cells (EPC) for enhancing bone growth in severe atrophic cases. Stem/progenitor cell-based regenerative therapy may prove to be the best option to meet individual patient needs and open new horizons in periodontal, maxillofacial and implant surgery.