Parathyroid hormone gene-activated matrix with DFDBA/collagen composite matrix enhances bone regeneration in rat calvarial bone defects.

BACKGROUND: Gene-activated matrix (GAM) induces sustained local production of growth factors to promote tissue regeneration. GAM contains a plasmid DNA (pDNA) encoding target proteins that is physically entrapped within a biodegradable matrix carrier. GAM with a pDNA encoding the first 34 amino acids of parathyroid hormone (PTH 1-34) and a collagen matrix enhances bone regeneration in long bone defects. Demineralized freeze-dried bone allograft (DFDBA) is a widely used osteoinductive bone graft. The present study determined the osteogenic effects of PTH-GAM with a collagen or DFDBA/collagen composite (D/C) matrix for treating craniofacial bone defects. METHODS: We constructed a pDNA encoding human PTH 1-34 and performed cyclic AMP ELISA to verify the bioactivity of PTH 1-34. Next, we generated a D/C matrix and PTH-GAMs containing a collagen matrix (PTH-C-GAM) or D/C matrix (PTH-D/C-GAM). Rats with critical-sized calvarial bone defects were divided into four groups, namely, untreated rats (sham group) and rats grafted with D/C matrix, PTH-C-GAM, or PTH-D/C-GAM (D/C, PTH-C-GAM, or PTH-D/C-GAM groups, respectively). PTH expression was determined by performing immunohistochemical staining after 4 and 8 weeks. New bone formation was evaluated by performing radiography, dual-energy X-ray absorptiometry, microcomputed tomography, and histological examination. RESULTS: PTH pDNA-transfected cells secreted bioactive PTH 1-34. Moreover, PTH was expressed at 4 and 8 weeks after the surgery in rats in the PTH-C-GAM group but not in rats in the D/C group. New bone formation in the calvarial bone defects, from more to less, was in the order of PTH-D/C-GAM, PTH-C-GAM, D/C, and sham groups. CONCLUSION: Our results indicate that PTH-GAM with a collagen matrix promotes local PTH production for at least 8 weeks and bone regeneration in craniofacial bone defect. Moreover, our results indicate that replacement of the collagen matrix with the D/C matrix improves the osteogenic effects of PTH-GAM.

Cell Therapy With G-CSF-Mobilized Stem Cells in a Rat Osteoarthritis Model.

G-CSF-mobilized peripheral blood stem cells (gm-PBSCs) offer a convenient cell source for treatment of hematopoietic and vascular disorders. Whether gm-PBSCs provide beneficial effects on skeleton diseases, such as osteoarthritis (OA), remains unknown. This study was undertaken to address the hypothesis that gm-PBSCs promote articular regeneration in OA. Here we studied the effect of single-dose intra-articular injection of gm-PBSCs from male donors delivered in hyaluronic acid (HA) on papain-induced OA in the knee joints of female Sprague-Dawley (SD) rats. Contralateral OA knee joints received single-dose HA alone and served as vehicle controls. We evaluated the histologic changes in glycosaminoglycan, type II collagen, type X collagen, modified Mankin score, and cell apoptosis rate in the articular cartilage of rat knees. We demonstrated that gm-PBSCs were mobilized to the peripheral blood via G-CSF infusion for 5 days in SD rats with increasing CD34(+) percentage up to 55-fold. We showed that gm-PBSCs inhibit progression of papain-induced OA via reducing articular surface irregularity, fibrillation, and erosion, preventing cellular necrosis and loss of chondrogenic proteins, such as glycosaminoglycan and type II collagen, at both 3 and 6 weeks after treatment. Moreover, gm-PBSCs reduced modified Mankin scores and cellular apoptosis rates compared with HA alone. Our findings demonstrate that HA plus gm-PBSCs, rather than HA alone, inhibits progression of OA in rats in vivo. Thus, intra-articular injection of gm-PBSCs is a convenient protocol for treating OA with consistent beneficial effects.

Hypoxic mesenchymal stem cells engraft and ameliorate limb ischaemia in allogeneic recipients.

AIMS: Local injection of stem cells or endothelial progenitors directly into the ischaemic tissue remains an option for the management of arterial occlusion. Bone marrow-derived mesenchymal stem cells (MSCs) represent a promising alternative autologous cell source for ischaemic limb cell therapy. However, methods for applying MSCs in allogeneic transplantation remain to be developed. The purpose of this study was to evaluate the therapeutic potential of MSCs cultured under a different environment in ameliorating limb ischaemia in allogeneic recipients. METHODS AND RESULTS: Here, we demonstrated that hypoxic MSCs from B6 mice ameliorate limb ischaemia of Balb/c mice compared with normoxic MSCs. We also demonstrated that hypoxic MSCs have an increased ability to engraft in allogeneic recipients by reducing natural killer (NK) cytotoxicity and decrease the accumulation of host-derived NK cells when transplanted in vivo. These allogeneic hypoxic MSCs gave rise to CD31+ endothelial cells and α-smooth muscle actin (SMA)+ and desmin+ muscle cells, thereby enhancing angiogenesis and restoring muscle structure. Moreover, application of anti-NK antibodies together with normoxic MSCs enhanced angiogenesis and prevented limb amputation in allogeneic recipients with limb ischaemia. CONCLUSION: These results strongly suggest that hypoxic MSCs are intrinsically immunoprivileged and can serve as a 'universal donor cell' for treating cardiovascular diseases.

Mesenchymal stem cells from a hypoxic culture improve and engraft Achilles tendon repair.

BACKGROUND: Bone marrow-derived mesenchymal stem cells (MSCs) from humans cultured under hypoxic conditions increase bone healing capacity. HYPOTHESIS: Rat MSCs cultured under hypoxic conditions increase the tendon healing potential after transplantation into injured Achilles tendons. STUDY DESIGN: Controlled laboratory study. METHODS: Biomechanical testing, histological analysis, and bromodeoxyuridine (BrdU) labeling/collagen immunohistochemistry were performed to demonstrate that augmentation of an Achilles tendon rupture site with hypoxic MSCs increases healing capacity compared with normoxic MSCs and controls. Fifty Sprague-Dawley rats were used for the experiments, with 2 rats as the source of bone marrow MSCs. The cut Achilles tendons in the rats were equally divided into 3 groups: hypoxic MSC, normoxic MSC, and nontreated (vehicle control). The uncut tendons served as normal uncut controls. Outcome measures included mechanical testing in 24 rats, histological analysis, and BrdU labeling/collagen immunohistochemistry in another 24 rats. RESULTS: The ultimate failure load in the hypoxic MSC group was significantly greater than that in the nontreated or normoxic MSC group at 2 weeks after incision (2.1 N/mm(2) vs 1.1 N/mm(2) or 1.9 N/mm(2), respectively) and at 4 weeks after incision (5.5 N/mm(2) vs 1.7 N/mm(2) or 2.7 N/mm(2), respectively). The ultimate failure load in the hypoxic MSC group at 4 weeks after incision (5.5 N/mm(2)) was close to but still significantly less than that of the uncut tendon (7.2 N/mm(2)). Histological analysis as determined by the semiquantitative Bonar histopathological grading scale revealed that the hypoxic MSC group underwent a significant improvement in Achilles tendon healing both at 2 and 4 weeks when compared with the nontreated or normoxic MSC group via statistical analysis. Immunohistochemistry further demonstrated that the hypoxic and normoxic MSC groups had stronger immunostaining for type I and type III collagen than did the nontreated group both at 2 and 4 weeks after incision. Moreover, BrdU labeling of MSCs before injection further determined the incorporation and retention of transplanted cells at the rupture site. CONCLUSION: Transplantation of hypoxic MSCs may be a better and more readily available treatment than normoxic MSCs for Achilles tendon ruptures. CLINICAL RELEVANCE: The present study provides evidence that transplantation of hypoxic MSCs may be a promising therapy for the treatment of Achilles tendon ruptures.

Efficient expansion of mesenchymal stem cells from mouse bone marrow under hypoxic conditions.

To realize the therapeutic potential of mesenchymal stem cells (MSCs), a large number of high-quality MSCs isolated from different species, such as mouse, were acquired for preclinical animal studies. Surprisingly, isolation and purification of mouse MSCs (mMSCs) is arduous because of the low frequency of MSCs and contamination of haematopoietic cells in culture. We have developed a method based on low density and hypoxic culture to isolate and expand mMSCs from different strains, including BALB/c, C57BL/6J, FVB/N and DBA/2. The cells from all of the strains expanded more rapidly when plated at low density in hypoxic culture compared with normoxic culture. These cells expressed CD44, CD105, CD29 and Sca-1 markers but not CD11b, CD34, CD45 and CD31 markers. Moreover, they were able to differentiate along osteoblastic, adipocytic and chondrocytic lineages. In conclusion, we have developed a robust method for isolation and expansion of mMSCs by combining low-density culture with hypoxic culture.

Benefits of hypoxic culture on bone marrow multipotent stromal cells.

Cultivation of cells is usually performed under atmospheric oxygen tension; however, such a condition does not replicate the hypoxic conditions of normal physiological or pathological status in the body. Recently, the effects of hypoxia on bone marrow multipotent stromal cells (MSCs) have been investigated. In a long-term culture, hypoxia can inhibit senescence, increase the proliferation rate and enhance differentiation potential along the different mesenchymal lineages. Hypoxia also modulates the paracrine effects of MSCs, causing upregulation of various secretable factors, including the vascular endothelial growth factor and interleukin-6, and thereby promoting wound healing and diabetic fracture healing. Finally, hypoxia plays an important role in mobilization and homing of MSCs, primarily by its ability to induce stromal cell-derived factor-1 expression along with its receptor, CXCR4. After transplantation, an ischemic environment, that is the combination of hypoxia and lack of nutrition, can lead to apoptosis or cell death, which can be overcome by the hypoxic preconditioning of MSCs and overexpression of prosurvival genes like Akt, HO-1 and Hsp70. This review emphasizes that hypoxia is an important factor in all major aspects of stem cell biology, and the mechanism involved in the hypoxic inducible factor-1signaling pathway behind these responses is also discussed.

Oct4 and Nanog directly regulate Dnmt1 to maintain self-renewal and undifferentiated state in mesenchymal stem cells.

The roles of Oct4 and Nanog in maintaining self-renewal and undifferentiated status of adult stem cells are unclear. Here, increase in Oct4 and Nanog expression along with increased proliferation and differentiation potential but decreased spontaneous differentiation were observed in early-passage (E), hypoxic culture (H), and p21 knockdown (p21KD) mesenchymal stem cells (MSCs) compared to late-passage (L), normoxic culture (N), and scrambled shRNA-overexpressed (Scr) MSCs. Knockdown of Oct4 and Nanog in E, H, and p21KD MSCs decreased proliferation and differentiation potential and enhanced spontaneous differentiation, whereas overexpression of Oct4 and Nanog in L, N, and Scr MSCs increased proliferation and differentiation potential and suppressed spontaneous differentiation. Oct4 and Nanog upregulate Dnmt1 through direct binding to its promoter, thereby leading to the repressed expression of p16 and p21 and genes associated with development and lineage differentiation. These data demonstrate the important roles of Oct4 and Nanog in maintaining MSC properties.

Scale-up of MSC under hypoxic conditions for allogeneic transplantation and enhancing bony regeneration in a rabbit calvarial defect model.

To realize the therapeutic potential of mesenchymal stem cells (MSCs), we aimed to develop a method for isolating and expanding New Zealand rabbit MSCs in a great scale. Rabbit MSCs expanded under hypoxic and normoxic conditions were compared in terms of replication capacity, differentiation potential, and the capacity for allogeneic transplantation in a calvarial defect model. The cells from all tested rabbits were expanded more rapidly when plated at low-density under hypoxic conditions compared to under normoxic conditions. Moreover, cells expanded under hypoxic conditions increased in the potential of osteoblastic, adipocytic, and chondrocytic differentiation. More importantly, radiographic analysis and micro-CT measurement of bone volume revealed the hypoxic cells when transplanted in the calvarial defects of another rabbit increased in the ability to repair bone defect compared to the normoxic cells. Six weeks after allogeneic transplantation of hypoxic MSCs, histological analysis revealed a callus spanned the length of the defect, and several bone tissues spotted in the implant. At 12 weeks, new bone had formed throughout the implant. Using BrdU labeling to track the transplanted cells, the hypoxic cells were more detected in the newly formed bone compared to the normoxic cells. For defects treated with allogeneic MSCs, no adverse host response could be detected at any time-point. In conclusion, we have developed a robust method for isolation and expansion of rabbit MSCs by combining low-density with hypoxic culture, which can be applied for the design of clinical trials in allogeneic transplantation of MSCs for bone healing.

Mesenchymal stem cell-conditioned medium facilitates angiogenesis and fracture healing in diabetic rats.

The most critical factor for fracture union is the blood supply to the fracture site, which is usually impaired in patients with diabetes. Recently, mesenchymal stem cells-derived conditioned medium (MSC-CM) has shown significantly higher levels of angiogenic factors, such as VEGF and IL-6. We demonstrate in this report that MSC-CM delivered in gelatin sponges stimulates angiogenesis and promotes fracture healing in a diabetic rat model. Subcutaneous implantation of gelatin sponges soaked in MSC-CM demonstrated better tissue ingrowth and higher capillary densities at 2 and 3 weeks than gelatin sponges in minimal essential medium (MEM) or 293 cell-derived conditioned medium (293-CM). Implantation of fibular defects with gelatin sponges soaked in MSC-CM enhanced bone ingrowth and fracture healing rates compared to 293-CM and MEM groups at 8 weeks. Micro-computed tomography analysis further indicated a higher new bone volume in the MSC-CM group compared to the other diabetic groups. Histological analysis with CD31 immunostaining also revealed that MSC-CM increased endothelial cell counts compared to the other groups. Together, these results indicated that gelatin sponges used to deliver MSC-CM promote angiogenesis and fracture healing in a diabetic model and may be an alternative strategy for treating fracture non-union in patients with diabetes.

Intramarrow bone morphogenetic protein 4 gene delivery enhances early implant stability in femurs of ovariectomized rabbits.

BACKGROUND: Sufficient early implant stability is critical to prevent excessive micromovement of the implant during peri-implant healing and to ensure the success of osseointegration. Implants placed in osteoporotic bones are often associated with low early implant stability. The purpose of this study is to determine the effects of intramarrow bone morphogenetic protein 4 (BMP4) gene delivery on early implant stability and peri-implant healing. METHODS: Adenoviruses encoding human BMP4 or LacZ were introduced into the femoral osteotomy sites immediately before implant placement in ovariectomized rabbits. The implant stability was determined by resonance frequency analysis at weeks 0, 4, and 8. Changes in cortical bone thickness and intrascrew bone formation at weeks 4 and 8 were evaluated by microcomputed tomography analysis and undecalcified histologic examination, respectively. RESULTS: Intramarrow BMP4 gene delivery resulted in more improvement in implant stability at both weeks 4 and 8. Increased increment in peri-implant cortical bone thickness and better intrascrew bone formation were found in the BMP4 group compared to the LacZ group. CONCLUSION: The results of this study suggest that intramarrow adenoviral gene delivery of BMP4 enhances peri-implant bone healing and improves early implant stability in osteoporotic rabbit femurs.

Knockdown of p21(Cip1/Waf1) enhances proliferation, the expression of stemness markers, and osteogenic potential in human mesenchymal stem cells.

Mammalian aging of many tissues is associated with a decline in the replicative and functional capacity of somatic stem cells. Understanding the basis of this decline is a major goal of aging research. Human bone marrow-derived multipotent stromal cells (MSCs) have been applied in the treatment of fracture nonunion. Clinical application of MSCs requires abundant cells that can be overcome by ex vivo expansion of cells, but often at the expense of stemness and differentiation potentiality. We first demonstrated that late-passage MSCs exhibited decreased proliferation capacity, reduced expression of stemness markers such as Oct-4 and Nanog, and deterioration of osteogenic potential. Further, late-passage MSCs showed increased expression of p21(Cip1/Waf1) (p21), an inhibitor of the cyclin-dependent kinase. Knockdown of p21 by lentivirus-mediated shRNAs against p21 in late-passage MSCs increased the proliferation capacity, the expression of Oct-4 and Nanog, and osteogenic potential compared with cells transduced with control shRNA. More importantly, reduction in p21 expression in MSCs enhanced the bone repair capacity of MSCs in a rodent calvarial defect model. Knockdown of p21 in MSCs also increased the telomerase activity and telomere length, and did not show chromosomal abnormalities or acquire transformation ability. Therefore, these data successfully demonstrate the involvement of senescence gene in the expression of stemness markers and osteogenic potential of MSCs.

Intramarrow bone morphogenetic protein 4 gene delivery improves local bone quality in femurs of ovariectomized rabbits.

BACKGROUND: Poor bone quality at implant recipient site is a major risk factor for implant failure. The purpose of this study is to examine the potential of intramarrow bone morphogenetic protein 4 (BMP4) gene delivery for local bone quality improvement. METHODS: Adenoviral vector encoding human BMP4 (Ad-BMP4) was constructed. Adenovirus encoding ß-galactosidase (Ad-LacZ) was used as a control virus. Ad-BMP4 and Ad-LacZ were injected into femurs of ovariectomized rabbits. The temporal changes in bone mineral density at injected areas were determined by repeated measurements by dual-energy x-ray absorptiometry at 0, 1, 2, 4, and 8 weeks after injection. The effects of gene delivery on cortical bone and cancellous bone were evaluated by microcomputed tomography analysis and histologic examination at 8 weeks. RESULTS: The bone mineral density of the BMP4 group was significantly higher than the LacZ group at 4 and 8 weeks by 61% and 35%, respectively. Results from microcomputed tomography analysis and histologic examination at 8 weeks indicated thicker cortical bone and denser cancellous bone in the BMP4 group compared to the LacZ group. CONCLUSIONS: Intramarrow gene delivery of BMP4 effectively improved local bone quality for at least 8 weeks. The sustained delivery of osteogenic factors via local gene therapy approach may reduce implant failures associated with poor local bone quality.

Enhancement of wound healing by human multipotent stromal cell conditioned medium: the paracrine factors and p38 MAPK activation.

Wound healing can be improved by transplanting mesenchymal stem cells (MSCs). In this study, we have demonstrated the benefits of the conditioned medium derived from human MSCs (CM-MSC) in wound healing using an excisional wound model. CM-MSC accelerated wound closure with increased reepithelialization, cell infiltration, granulation formation, and angiogenesis. Notably, CM-MSC enhanced epithelial and endothelial cell migration, suggesting the contribution of increased cell migration to wound healing enhanced by CM-MSC. Cytokine array, ELISA analysis, and quantitative RT-PCR revealed high levels of IL-6 in CM-MSC. Moreover, IL-6 added to the preconditioned medium enhanced both cell migration and wound healing, and antibodies against IL-6 blocked the increase in cell motility and wound closure by CM-MSC. The IL-6 secretory pathway of MSCs was inhibited by SB203580, an inhibitor of p38 MAPK or siRNA against p38 MAPK, suggesting IL-6 secretion by MSCs is mediated through the activation of p38 MAPK. Inactivation of p38 MAPK also reduced the expression and production of IL-8 and CXCL1 by MSCs, both of which were also demonstrated to enhance cell migration and wound closure. Thus, our data suggest MSCs promote wound healing through releasing a repertoire of paracrine factors via activation of p38 MAPK, and the CM-MSC may be applied to enhance wound healing.

Hypoxia inhibits senescence and maintains mesenchymal stem cell properties through down-regulation of E2A-p21 by HIF-TWIST.

Although low-density culture provides an efficient method for rapid expansion of human mesenchymal stem cells (MSCs), MSCs enriched by this method undergo senescence and lose their stem cell properties, which could be preserved by combining low-density and hypoxic culture. The mechanism was mediated through direct down-regulation of E2A-p21 by the hypoxia-inducible factor-1α (HIF-1α)-TWIST axis. Expansion under normoxia induced E2A and p21 expression, which were abrogated by overexpression of TWIST, whereas siRNA against TWIST up-regulated E2A and p21 in hypoxic cells. Furthermore, siRNA against p21 in normoxic cells enhanced proliferation and increased differentiation potential, whereas overexpression of p21 in hypoxic cells induced a decrease in proliferation and a loss of differentiation capacity. More importantly, MSCs expanded under hypoxic conditions by up to 100 population doublings, exhibited telomerase activity with maintained telomere length, normal karyotyping, and intact genetic integrity, and did not form tumors. These results support low-density hypoxic culture as a method for efficiently expanding MSCs without losing stem cell properties or increasing tumorigenicity.