Pigment epithelium-derived factor (PEDF) reduced expression and synthesis of SOST/sclerostin in bone explant cultures: implication of PEDF-osteocyte gene regulation in vivo.

Mutations in Serpinf1 gene which encodes pigment epithelium-derived factor (PEDF) lead to osteogenesis imperfecta type VI whose hallmark is defective matrix mineralization. We reported previously that PEDF reduced expression and synthesis of Sost/Sclerostin as well as other osteocytes genes encoding proteins that regulate matrix mineralization [1]. To determine whether PEDF had an effect on osteocyte gene expression in bone, we used bone explant cultures. First, osteocytes were isolated from surgical waste of bone fragments obtained from patients undergoing elective foot surgeries under approved IRB protocol by Penn State College of Medicine IRB committee. Primary osteocytes treated with PEDF reduced expression and synthesis of Sost/Sclerostin and matrix phosphoglycoprotein (MEPE) as well as dentin matrix protein (DMP-1). On the whole, PEDF reduced osteocyte protein synthesis by 50% and by 75% on mRNA levels. For bone explants, following collagenase digestion, bone fragments were incubated in alpha-MEM supplemented with 250 ng/ml of PEDF or BSA. After 7 days of incubation in a medium supplemented with PEDF, analysis of mRNA by PCR and protein by western blotting of encoded osteocyte proteins showed reduced Sclerostin synthesis by 39% and MEPE by 27% when compared to fragments incubated in medium supplemented with BSA. mRNA expression levels of osteocytes in bone fragments treated with PEDF were reduced by 50% for both SOST and MEPE when compared to BSA-treated bone fragments. Taken together, the data indicate that PEDF has an effect on osteocyte gene expression in bone and encourage further studies to examine effect of PEDF on bone formation indices in animal models and its effect on osteocyte gene expression in vivo following PEDF administration.

Pigment epithelium derived factor regulates human Sost/Sclerostin and other osteocyte gene expression via the receptor and induction of Erk/GSK-3beta/beta-catenin signaling.

Mutations in Serpinf1 gene which encodes pigment epithelium derived factor (PEDF) lead to osteogenesis imperfecta type VI whose hallmark is defective mineralization. We reported that PEDF suppressed expression of Sost/Sclerostin and other osteocyte related genes in mineralizing osteoblast cultures and suggested that this could be part of the mechanisms by which PEDF regulates matrix mineralization (Li et al. J Cellular Phys. 2014). We have used a long-term differentiated mineralizing osteoblast culture (LTD) to define mechanisms by which PEDF regulates osteocyte gene expression. LTD cultures were established by culturing human osteoblasts in an osteogenic medium for 4 months followed by analysis of osteocytes related genes and encoded proteins. LTD cells synthesized Sclerostin, matrix extracellular phosphoglycoprotein (MEPE) and dentin matrix protein (DMP-1) and their synthesis was reduced by treatment with PEDF. Treatment of the cultures with PEDF induced phosphorylation of Erk and glycogen synthase kinase 3-beta (GSK-3ß), and accumulation of nonphosphorylated ß-catenin. Inhibition of Erk activation and neutralizing antibodies to the pigment epithelium derived receptor (PEDF-R) suppressed GSK-3ß phosphorylation and accumulation of nonphosphorylated ß-catenin in presence of PEDF. Topflash assays demonstrated that PEDF activated luciferase reporter activity and this activity was inhibited by treatment with Erk inhibitor or neutralizing antibodies to PEDF-R. Dickkopf-related protein 1 treatment of the cells in presence of PEDF had minimal effect suggesting that GSK-3ß phosphorylation and accumulation of nonphosphorylayted ß-catenin may not involve LRP5/6 in osteocytes. Taken together, the data demonstrate that PEDF regulates osteocyte gene expression through its receptor and possible involvement of Erk/GSK-3ß/ß-catenin signaling pathway.

Pigment epithelium derived factor upregulates expression of vascular endothelial growth factor by human mesenchymal stem cells: Possible role in PEDF regulated matrix mineralization.

Pigment epithelium-derived factor (PEDF) encoded by serpinf1 is a potent antiangiogenic factor found in a wide variety of fetal and adult tissues. Several reports have shown that lack of PEDF leads to osteogenesis imperfecta (OI) type VI whose hallmark is a defect in mineralization that leads to excessive osteoid build up that fails to mineralize. Because PEDF is antiangiogenic factor it would pose serious consequences on bone development and healing of fractures. To understand possible mechanisms by which PEDF plays a role in bone development and regulation of matrix mineralization, we determined the effects of exogenous PEDF on vascular endothelial growth factor (VEGF) expression by human mesenchymal stem cells (hMSCs) and mechanisms of its regulation by PEDF. Human MSCs incubated in normal medium supplemented with exogenous PEDF increased VEGF expression; this increase was also seen when PEDF was added to hMSCs undergoing osteogenic differentiation. MSCs maintained in osteogenic medium increased synthesis of both VEGF and PEDF but both factors were maintained relatively in balance during differentiation. To understand mechanisms by which exogenous PEDF regulated VEGF expression, hMSCs exposed to PEDF activated Erk signaling pathway in MSCs; inhibition of Erk signaling reduced VEGF mRNA expression as well as protein production suggesting that PEDF regulates VEGF expression in MSCs via Erk signaling pathway. In conclusion, PEDF increases VEGF expression by MSCs suggesting that regulation of VEGF by PEDF may be part of the mechanisms by which PEDF regulates osteoblastic mineralization.

Engraftability of Murine Bone Marrow-Derived Multipotent Mesenchymal Stem Cell Subpopulations in the Tissues of Developing Mice following Systemic Transplantation.

INTRODUCTION: Cell therapies for generalized musculoskeletal diseases would require distribution of cells to all the skeletal tissues; however, there are controversies regarding the transplantability of multipotent mesenchymal stems cells (MSCs). We generated single-cell subpopulations of MSCs from murine bone marrow and assessed them for differences in trafficking through the circulatory system and engraftment in bone and other tissues. MATERIALS AND METHODS: Seven single-cell clonal subpopulations were generated by serial dilution of GFP-marked MSCs isolated from bone marrow. The subpopulations were examined for putative MSC surface marker expression, in vitro differentiation toward osteogenic and adipogenic lineages, migration and engraftment in different tissues following intravenous delivery in normal, sublethally irradiated neonatal mice. RESULTS: The surface marker expression profile revealed notable differences among clonal cells, specifically CD44 and CD105. All the cell subpopulations differentiated toward osteogenic and adipogenic lineages, with some committed to only one or the other. Two clones enriched in CXCR4 expression were highly efficient in migrating and engrafting in skeletal tissue including bone; this confirmed the role of this chemokine in cell migration. Donor cells retrieved from various tissues displayed different morphologies and potential differentiation into tissue cell type of engraftment, suggesting modification by the tissues in which the donor cells engrafted. CONCLUSION: We have reported that, within bone marrow, there are heterogeneous subpopulations of MSCs that may differ in their ability to migrate in the circulatory system and engraft in different tissues.

Pigment epithelium derived factor suppresses expression of Sost/Sclerostin by osteocytes: implication for its role in bone matrix mineralization.

Mutations in Serpinf1 gene which encodes pigment epithelium derived factor (PEDF) lead to osteogenesis imperfecta type VI whose hallmark is defective mineralization. Mechanisms by which PEDF regulates matrix mineralization remain unknown. We examined effect of exogenous PEDF on expression of osteoblastic and osteocytic related genes and proteins in mineralizing osteoblast culture. Mineralizing human osteoblasts supplemented with exogenous PEDF for 14 days deposited 47% more mineral than cells cultured without PEDF. Analysis of selected gene expression by cells in mineralizing cultures supplemented with exogenous PEDF showed reduction in expression of Sclerostin (Sost) by 70%, matrix extracellular phosphoglycoprotein (MEPE) by 75% and dentin matrix protein (DMP-1) by 20% at day 14 of culture. Phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX) expression was not affected. Western blotting and immunoprecipitation showed that sclerostin and MEPE synthesis by osteocytes were reduced by 50% and 60% respectively in mineralizing osteoblasts containing exogenous PEDF. Primary osteocytes exposed to PEDF also reduced synthesis of Sost/sclerostin by 50% within 24 h. For osteoblastic genes, Bone sialoprotein (BSP) was expressed at 75% higher by day 7 in cultures containing exogenous PEDF while Col1A1 expression remained high at all-time points. Total beta-catenin was increased in mineralizing osteoblastic cells suggesting increased Wnt activity. Taken together, the data indicate that PEDF suppressed expression of factors that inhibit mineralization while enhancing those that promote mineralization. The findings also suggest that PEDF may regulate Sost expression by osteocytes leading to enhanced osteoblastic differentiation and increased matrix mineralization.

Multipotent mesenchymal stem cells from human subacromial bursa: potential for cell based tendon tissue engineering.

Rotator cuff injuries are a common clinical problem either as a result of overuse or aging. Biological approaches to tendon repair that involve use of scaffolding materials or cell-based approaches are currently being investigated. The cell-based approaches are focused on applying multipotent mesenchymal stem cells (MSCs) mostly harvested from bone marrow. In the present study, we focused on characterizing cells harvested from tissues associated with rotator cuff tendons based on an assumption that these cells would be more appropriate for tendon repair. We isolated MSCs from bursa tissue associated with rotator cuff tendons and characterized them for multilineage differentiation in vitro and in vivo. Human bursa was obtained from patients undergoing rotator cuff surgery and cells within were isolated using collagenase and dispase digestion. The cells isolated from the tissues were characterized for osteoblastic, adipogenic, chondrogenic, and tenogenic differentiation in vitro and in vivo. The results showed that the cells isolated from bursa tissue exhibited MSCs characteristics as evidenced by the expression of putative cell surface markers attributed to MSCs. The cells exhibited high proliferative capacity and differentiated toward cells of mesenchymal lineages with high efficiency. Bursa-derived cells expressed markers of tenocytes when treated with bone morphogenetic protein-12 (BMP-12) and assumed aligned morphology in culture. Bursa cells pretreated with BMP-12 and seeded in ceramic scaffolds formed extensive bone, as well as tendon-like tissue in vivo. Bone formation was demonstrated by histological analysis and immunofluorescence for DMP-1 in tissue sections made from the scaffolds seeded with the cells. Tendon-like tissue formed in vivo consisted of parallel collagen fibres typical of tendon tissues. Bursa-derived cells also formed a fibrocartilagenous tissue in the ceramic scaffolds. Taken together, the results demonstrate a new source of MSCs with a high potential for application in tendon repair.

Pigment epithelium-derived factor enhances differentiation and mineral deposition of human mesenchymal stem cells.

Pigment epithelium-derived factor (PEDF) is a potent antiangiogenic factor found in a wide variety of tissues. Recent findings indicated that lack of PEDF leads to osteogenesis imperfecta type VI whose hallmark is a defect in mineralization. We investigated the effects of PEDF on human mesenchymal stem cells (hMSCs) and signaling pathways through which PEDF displays its activities in hMSCs. hMSCs incubated in a medium supplemented with PEDF induced expression of osteoblastic-related genes. In addition, PEDF induced alkaline phosphatase (ALP) activity in MSCs at 14 days of incubation in maintenance medium; hMSCs incubated in osteogenic medium in presence of PEDF expressed 19% more ALP activity (35.655 ± 1.827 U/mg protein, p = .041 than cells incubated in the same medium without PEDF supplementation (29.956 ± 2.100 U/µg protein). hMSCs incubated in osteogenic medium in presence of PEDF deposited 50% more mineral (2.108 ± 0.306 OD/ml per well per 1 × 10(4) cells per square centimeter, p = .017) than MSCs incubated in absence of the protein (1.398 ± 0.098 OD/ml per well per 1 × 10(4) cells per square centimeter) as determined by Alizarin Red quantitation. Reduction in PEDF expression in MSCs by siRNA led to decreased ALP activity (33.552 ± 2.009 U/ng protein of knockdown group vs. 39.269 ± 3.533 U/ng protein of scrambled siRNA group, p = .039) and significant reduction in mineral deposition (0.654 ± 0.050 OD/ml per well per 1 × 10(4) cells per square centimeter of knockdown group vs. 1.152 ± 0.132 OD/ml per well per 1 × 10(4) cells per square centimeter of wild-type group, p = .010). Decreased ALP activity and mineral deposition were restored by supplementation with exogenous PEDF protein. PEDF activated ERK and AKT signaling pathways in MSCs to induce expression of osteoblastic-related genes. These data suggest that PEDF is involved in MSCs osteoblastic differentiation.

Cells derived from murine induced pluripotent stem cells (iPSC) by treatment with members of TGF-beta family give rise to osteoblasts differentiation and form bone in vivo.

BACKGROUND: Induced pluripotent stem cells (iPSC) are generated by reprogramming somatic cells into embryonic like state (ESC) using defined factors. There is great interest in these cells because of their potential for application in regenerative medicine. RESULTS: iPSC reprogrammed from murine tail tip fibroblasts were exposed to retinoic acid alone (RA) or in combination with TGF-ß1 and 3, basic fibroblast growth factor (bFGF) or bone morphogenetic protein 2 (BMP-2). The resulting cells expressed selected putative mesenchymal stem cells (MSCs) markers; differentiated toward osteoblasts and adipocytic cell lineages in vitro at varying degrees. TGF-beta1 and 3 derived-cells possessed higher potential to give rise to osteoblasts than bFGF or BMP-2 derived-cells while BMP-2 derived cells exhibited a higher potential to differentiate toward adipocytic lineage. TGF-ß1 in combination with RA derived-cells seeded onto HA/TCP ceramics and implanted in mice deposited typical bone. Immunofluorescence staining for bone specific proteins in cell seeded scaffolds tissue sections confirmed differentiation of the cells into osteoblasts in vivo. CONCLUSIONS: The results demonstrate that TGF-beta family of proteins could potentially be used to generate murine iPSC derived-cells with potential for osteoblasts differentiation and bone formation in vivo and thus for application in musculoskeletal tissue repair and regeneration.

Differentiating multipotent mesenchymal stromal cells generate factors that exert paracrine activities on exogenous MSCs: Implications for paracrine activities in bone regeneration.

The mechanisms by which multipotent mesenchymal stromal cells (MSCs) contribute to tissue repair following transplantation into host tissues remains poorly understood. Current concepts suggest that, in addition to differentiation into cells of the host tissues, MSCs also generate trophic factors that modulate host tissue microenvironment to aid in the repair process. In this communication, we assessed whether factors secreted by MSCs undergoing osteogenic differentiation induce expression of osteoblast markers in exogenous MSCs as well as their migration. Murine MSCs were cultured in osteogenic medium, and at different time points, medium conditioned by the cells was collected and assessed for its effects on differentiation and migration of exogenous MSCs. In addition, we determined whether MSCs infused into mice femurs expressed genes encoding for factors predicted to play a role in paracrine activities. The results showed that MSCs maintained in osteogenic medium, secreted factors at specific time points that induced alkaline phosphatase activity (ALP) in exogenous MSCs as well as their migration. MSCs infused into mice femurs and retrieved at different days expressed genes that encoded predicted factors that play a role in cell differentiation and migration. Neutralizing antibodies to bone morphogenetic protein-2 (BMP-2) led to the decrease in ALP activity by exogenous MSCs. These data demonstrated that, as MSCs differentiate toward osteogenic lineage, they secrete factors that induce recruitment and differentiation of endogenous progenitors. These data reveal mechanisms by which donor MSCs may contribute to the bone reparative process and provide a platform for designing approaches for stem cell therapies of musculoskeletal disorders.

Breast and abdominal adipose multipotent mesenchymal stromal cells and stage-specific embryonic antigen 4 expression.

We assessed human mesenchymal stem cells (MSCs) harvested from breast and abdominal adipose tissues enriched in embryonic stage-specific antigen (SSEA-4) expression for osteogenic and adipogenic differentiation in comparison to a mixed cell population. Human adipose was obtained from abdominal and breast tissues of females undergoing gastric bypass and breast reduction, respectively. SSEA-4-expressing cells were enriched from the mixed cell population by magnetic cell sorting and expanded in culture. The results showed that freshly isolated cells from breast and abdominal tissues based on adipose from 3 patients comprised 12 and 10% SSEA-4+ cells, respectively. At passage 0, 48% of the cells from breast adipose tissue were positive for SSEA-4 while 12% of the cells from abdominal adipose tissue were positive for this antigen. The level of SSEA-4-expressing cells remained relatively constant with passaging; SSEA-4-expressing cells from breast tissue comprised 45% of the total while 27% of the cells from abdominal adipose tissue expressed SSEA-4 at passage 5. Cells sorted for SSEA-4 expression exhibited a higher potential for differentiation toward osteogenic and adipogenc cell lineages in vitro when compared to a mixed population. Interestingly, SSEA-4 expression was lost upon differentiation, suggesting that the antigen marks a subpopulation of MSCs. Taken together, the data demonstrate that breast adipose tissue is highly enriched in a subpopulation of MSCs expressing SSEA-4 and suggest that SSEA-4 may be a marker of a subpopulation of MSCs with high potential for osteogenic and adipogenic differentiation.

Bone marrow stromal cells contribute to bone formation following infusion into femoral cavities of a mouse model of osteogenesis imperfecta.

Currently, there are conflicting data in literature regarding contribution of bone marrow stromal cells (BMSCs) to bone formation when the cells are systemically delivered in recipient animals. To understand if BMSCs contribute to bone cell phenotype and bone formation in osteogenesis imperfecta bones (OI), MSCs marked with GFP were directly infused into the femurs of a mouse model of OI (oim). The contribution of the cells to the cell phenotype and bone formation was assessed by histology, immunohistochemistry and biomechanical loading of recipient bones. Two weeks following infusion of BMSCs, histological examination of the recipient femurs demonstrated presence of new bone when compared to femurs injected with saline which showed little or no bone formation. The new bone contained few donor cells as demonstrated by GFP fluorescence. At 6 weeks following cell injection, new bone was still detectable in the recipient femurs but was enhanced by injection of the cells suspended in pepsin solubilized type I collagen. Immunofluorescence and immunohistochemical staining showed that donor GFP positive cells in the new bone were localized with osteocalcin expressing cells suggesting that the cells differentiated into osteoblasts in vivo. Biomechanical loading to failure in three point bending, revealed that, femurs infused with BMSCs in PBS or in soluble type I collagen were biomechanically stronger than those injected with PBS or type I collagen alone. Taken together, the results indicate that transplanted cells differentiated into osteoblasts in vivo and contributed to bone formation in vivo; we also speculate that donor cells induced differentiation or recruitment of endogenous cells to initiate reparative process at early stages following transplantation.

Low oxygen tension during incubation periods of chondrocyte expansion is sufficient to enhance postexpansion chondrogenesis.

To determine whether low oxygen (O(2)) tension during expansion affects the matrix density, as well as quantity, of cartilage formed, and to determine whether application of low O(2) tension during incubation periods alone is sufficient to modulate chondrogenic expression, rabbit chondrocytes expanded at either 21% O(2) or 5% O(2) were analyzed for glycosaminoglycan (GAG) and DNA content, total collagen, and gene expression during expansion and postexpansion aggregate cultures. When cultured as aggregates at 21% O(2), chondrocytes expanded at 5% O(2) produced cartilage aggregates that contained more total GAG, GAG per wet weight, GAG per DNA, and total collagen than chondrocytes expanded at 21% O(2). Less of an effect on GAG and collagen content was observed when aggregate culture was performed at 5% O(2). Real-time polymerase chain reaction analysis of COL2A1 expression showed upregulated levels of type IIA (an early marker) and IIB (a late marker) during expansion and elevated levels of type IIB during aggregate culture in chondrocytes expanded in low O(2). The application of low O(2) tension during incubation periods of chondrocyte expansion enhances the ultimate cartilage matrix density and quantity, and this enhancement can be achieved through the use of an O(2) control incubator.

Derivation of murine induced pluripotent stem cells (iPS) and assessment of their differentiation toward osteogenic lineage.

Induced pluripotent stem cells (iPSCs) have generated hope and excitement because of the potential they possess for generating patient-specific embryonic-like stem cells (ESCs). Although many hurdles remain to be solved before the cells can be applied clinically; studies directed toward understanding factors that control differentiation of the cells toward various cell lineages are prerequisites for their future application. In the present study, we generated murine iPSC and assessed their differentiation toward osteogenic lineage. Murine tail tip fibroblasts were reprogrammed into embryonic-like state by transduction with defined factors (Oct3/4, Sox2, c-Myc, and klf4) carried in a retroviral vector. The reprogrammed cells expressed ESC markers, gave rise to three germ layers as demonstrated by teratoma formation and immunofluorescence staining. These data confirmed that the reprogrammed cells exhibited ESC-like state. Treatment of iPSCs-derived embryoid bodies (EBs) with transforming growth factor beta 1 (TGF-beta1) in the presence of retinoic acid enhanced generation of MSC-like cells. The MSCs-like cells expressed putative makers associated with MSCs; the cells deposited calcium in vitro when cultured in osteogenic medium. Interestingly MSCs-like cells generated from iPSC directed EBs by treatment with retinoic acid and TGF-beta1 deposited more calcium in vitro than cells derived without TGF-beta1 treatment. Taken together, the data demonstrate that iPSC give rise to MSCs-like state and that the cells have potential to differentiate toward osteoblasts. In addition, brief treatment of iPSC-derived EBs with TGF-beta1 may be an approach for directing iPSC toward MSC-like state.

Effects of Culturing on the Stability of the Putative Murine Adipose Derived Stem Cells Markers.

Mesenchymal stem cells have generated much interest because of their potential use in regenerative medicine. The major draw back in the application of these cells is that there is no single marker or markers that have been established to identify and aid in isolating the cells from a variety of other cell types. The commonly expressed mesenchymal stem cell surface antigens include CD44, CD73, CD90.2, CD105, and CD146. In the present study we examined the stability of these surface antigens in culture and their potential application in identifying and isolating murine derived adipose derived stem cells. The data showed that the expression of these markers increased with culturing and appeared to stabilize by passage 8; the cells were sorted positively for the surface markers at this passage. Each subset was maintained in culture and evaluated for differentiation toward osteogenic lineage in vitro and in vivo. The CD73 and CD105 positive cell subsets demonstrated robust differentiation toward osteogenic lineage in vitro; the CD90.2+ cell subset exhibited the least differentiation toward osteogenic lineage. Assessment of the cell subpopulations for in vivo differentiation demonstrated that all the cell subsets exhibited potential to differentiate into osteoblasts. Taken together, these data suggest that this panel of markers although useful in identifying cells with potential to differentiate toward osteogenic lineage, cannot prospectively be used for enriching for ADSC from a variety of other cell types.

Potential implications of cell therapy for osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a brittle-bone disease whose hallmark is bone fragility. Since the disease is genetic, there is currently no available cure. Several pharmacological agents have been tried with not much success, except the recent use of bisphosphonates. Stem cells have been suggested as an alternative OI treatment, but many hurdles remain before this technology can be applied for treating patients with OI. This review summarizes what is known at present regarding the application of stem cells to treat OI using animal models, clinical trials using mesenchymal stem cells to treat patients with OI and the knowledge gained from the clinical trials. Application of gene therapy in combination with stem cells is also discussed. The hurdles to be overcome to bring stem cells close to the clinic and future perspectives are discussed.

Distribution of murine adipose-derived mesenchymal stem cells in vivo following transplantation in developing mice.

Systemic delivery of mesenchymal stem cells (MSCs) or stromal cells in vivo is attractive because it offers means of disseminating therapeutic cells to various tissues and organs in vivo. In the present study, we investigated the distribution and engraftment of the murine adipose-derived mesenchymal stem cells (ADSCs) without exposure to or exposed to bone microenvironment or transforming growth factor-beta1 (TGF-beta1) prior to transplantation into developing mice. The ADSCs harvested from the murine inguinal fat pad exhibited potential for differentiation toward osteogenic and adipogenic cell lineages in vitro. Fourteen days after systemic transplantation of the ADSCs marked with enhanced green fluorescent protein (EGFP) into developing mice, minimal donor GFP(+) cells were detected in the skeletal tissues in a limited number of the recipient mice. Exposure of the ADSCs to bone microenvironment for 7 or 14 days prior to transplantation into developing mice enhanced their migration and survival in the bones of the recipient mice. Exposure of ADSCs to TGF-beta1 prior to systemic transplantation exerted similar effects on cell migration and engraftment in various tissues, including the bones of the recipient developing mice. At 28 days following systemic transplantation, the ADSCs exposed to bone microenvironment were restricted mostly to the skeletal tissues of the recipient mice. Donor cells retrieved from the bones of the recipient mice at 28 days following cell transplantation expressed the differentiation markers Runx2 and Osterix (Osx). These data suggest that exposure of ADSCs to bone microenvironment or to TGF-beta1 prior to transplantation enhances their survival in the skeletal tissues following transplantation.

The effects of recombinant human bone morphogenetic protein-2, recombinant human bone morphogenetic protein-12, and adenoviral bone morphogenetic protein-12 on matrix synthesis in human annulus fibrosis and nucleus pulposus cells.

BACKGROUND CONTEXT: Bone morphogenetic proteins (BMPs) are potential therapeutic factors for degenerative discs, and BMP-12 does not have the osteogenic potential of BMP-2, making it better suited for intradiscal injection. However, no reports have compared the actions of BMP-2 and -12 on human annulus fibrosus (AF) and nucleus pulposus (NP) cells nor evaluated adenoviral-mediated gene therapy in human AF cells. PURPOSE: To evaluate and compare the effects of recombinant human (rh) BMP-2, rhBMP-12, and adenoviral BMP-12 (Ad-BMP-12) on nucleus pulposus and annulus fibrosis cell matrix protein synthesis. STUDY DESIGN: In vitro study using rhBMP-2 and -12 and adenoviral BMP-12 with human intervertebral disc (IVD) cells. METHODS: Human NP and AF IVD cells were isolated, maintained in monolayer, and incubated with BMP-2 or -12 for 2 days. AF and NP cells were transduced with Ad-BMP-12, pellets formed, and incubated for 6 days. Growth factor-treated cells were labelled with either 35-S or 3H-proline to assay matrix protein synthesis. RESULTS: rhBMP-2 increased NP proteoglycan, collagen, and noncollagen protein synthesis to 355%, 388%, and 234% of control. RhBMP-12 increased the same NP matrix proteins' synthesis to 140%, 143%, and 160% of control. Effects on AF matrix protein synthesis were minimal. Ad-BMP-12 significantly increased matrix protein synthesis and DNA content of AF and NP cells in pellet culture. NP synthesis of all matrix proteins and AF synthesis of proteoglycans was increased when the data were normalized to pellet DNA. AF synthesis of noncollagen protein and collagen was not modulated by Ad-BMP-12 if the data are normalized to pellet DNA content. CONCLUSIONS: Both rhBMP-2 and -12 increase human NP cell matrix protein synthesis while having minimal effects on AF cells. However, Ad-BMP-12 did increase matrix protein synthesis in both NP and AF cells, making it a potential therapy for enhancing matrix production in the IVD. These responses plus the proliferative action of Ad-BMP-12 seen in the current studies, and the lack of an osteogenic action noted in other studies justifies future studies to determine if gene therapy with BMP-12 could provide protective and/or reparative actions in degenerating discs.

Distribution of single-cell expanded marrow derived progenitors in a developing mouse model of osteogenesis imperfecta following systemic transplantation.

We evaluated single-cell-expanded, marrow-derived progenitors for engraftment in a developing mouse model of osteogenesis imperfecta (OI) following systemic transplantation. The present study was initiated to evaluate the potential of mesenchymal stem cells to treat OI. Single-cell-derived progenitors were prepared from marrow stromal cells harvested from normal mice. Selected single-cell-expanded progenitors marked with green fluorescent protein were injected into the neonatal mouse model of OI, and the recipient mice were sacrificed at 2 and 4 weeks following cell transplantation. Examination of the tissues harvested from recipient mice at 2 and 4 weeks after cell transplantation demonstrated that the cells extravasated and engrafted in most of the bones as well as other tissues. Tissue sections made from the tibias and femurs of a selected recipient mouse showed that the cells were distributed in bone marrow, trabecular, and cortical bone as demonstrated by histology and confocal microscopy. The cells that engrafted in the bones of the recipient mouse synthesized and deposited type I collagen composed of alpha1(I) and alpha2(I) collagen heterotrimers. Genotyping and gene expression analysis of the cells retrieved from the bones of the recipient mouse at 2 and 4 weeks demonstrated that the cells expressed osteoblast-specific genes, suggesting that the donor cells differentiated into osteoblasts in vivo with no evidence of cell fusion. These data suggest that progenitors infused in developing mice will engraft in various tissues including bones, undergo differentiation, and deposit matrix and form bone in vivo. Disclosure of potential conflicts of interest is found at the end of this article.

Cartilage tissue engineering for laryngotracheal reconstruction: comparison of chondrocytes from three anatomic locations in the rabbit.

Tissue engineering may provide a technique to generate cartilage grafts for laryngotracheal reconstruction in children. The present study used a rabbit model to characterize cartilage generated by a candidate tissue engineering approach to determine, under baseline conditions, which chondrocytes in the rabbit produce tissue-engineered cartilage suitable for in vivo testing in laryngotracheal reconstruction. We characterized tissue-engineered cartilage generated in perfused bioreactor chambers from three sources of rabbit chondrocytes: articular, auricular, and nasal cartilage. Biomechanical testing and histological, immunohistochemical, and biochemical assays were performed to determine equilibrium unconfined compression (Young's) modulus, and biochemical composition and structure. We found that cartilage samples generated from articular or nasal chondrocytes lacked the mechanical integrity and stiffness necessary for completion of the biomechanical testing, but five of six auricular samples completed the biomechanical testing (moduli of 210 +/- 93 kPa in two samples at 3 weeks and 100 +/- 65 kPa in three samples at 6 weeks). Auricular samples showed more consistent staining for proteoglycans and collagen II and had significantly higher glycosaminoglycan (GAG) content and concentration and higher collagen content than articular or nasal samples. In addition, the delayed gadolinium enhanced MRI of cartilage (dGEMRIC) method revealed variations in GAG spatial distribution in auricular samples that were not present in articular or nasal samples. The results indicate that, for the candidate tissue engineering approach under baseline conditions, only rabbit auricular chondrocytes produce tissue-engineered cartilage suitable for in vivo testing in laryngotracheal reconstruction. The results also suggest that this and similar tissue engineering approaches must be optimized for each potential source of chondrocytes.

Progenitors systemically transplanted into neonatal mice localize to areas of active bone formation in vivo: implications of cell therapy for skeletal diseases.

The potential of cell or gene therapy to treat skeletal diseases was evaluated through analysis of transplanted osteoprogenitors into neonatal homozygous and heterozygous osteogenesis imperfecta mice (oim). The osteoprogenitors used for transplantation were prepared by injection of mesenchymal stem cells (MSCs) marked with the green fluorescent protein (GFP) into normal mice with the subsequent retrieval of the cells at 35 days. The retrieved cells referred to here as osteoprogenitors were expanded in culture and transplanted into the 2-day-old oim mice via the superficial temporal vein. The recipient mice were evaluated at 2 and 4 weeks after cell transplantation. Four weeks after transplantation, tissue sections made from femurs and tibias of oim mice showed that the GFP-positive (GFP(+)) cells were distributed on the surfaces of the bone spicules in the spongiosa, the area of active bone formation. In the diaphysis, the GFP(+) cells were distributed in the bone marrow, on the endosteal surfaces, and also in the cortical bone. Immunofluorescence localization for GFP confirmed that the fluorescence seen in tissue sections was due to the engrafted donor cells, not bone autofluorescence. Gene expression analysis by polymerase chain reaction of the GFP(+) cells retrieved from the bones and marrow of the recipient mice demonstrated that the cells from bone were osteoblasts, whereas those from bone marrow were progenitors. These data demonstrate that MSCs delivered systemically to developing osteogenesis imperfecta mice engraft in bones, localize to areas of active bone formation, differentiate into osteoblasts in vivo, and may contribute to bone formation in vivo.