Mesenchymal stem cell expression of stromal cell‐derived factor‐1β augments bone formation in a model of local regenerative therapy

Bone has the potential for spontaneous healing. However, this process often fails in patients with co‐morbidities requiring clinical intervention. Numerous studies have revealed that bone marrow‐derived mesenchymal stem/stromal cells (BMSCs) hold great potential for regenerative therapies. Common problems include poor cell engraftment, which can be addressed by irradiation prior to transplantation. Increasing evidence suggests that stromal cell‐derived factor‐1 (SDF‐1) is involved in bone formation. However, osteogenic contributions of the beta splice variant of SDF‐1 (SDF‐1β), which is highly expressed in bone, remain unclear. Using the tetracycline (Tet)‐regulatory system we have shown that SDF‐1β enhances BMSC osteogenic differentiation in vitro. Here we test the hypothesis that SDF‐1β augments bone formation in vivo in a model of local BMSC transplantation following irradiation. We found that SDF‐1β, expressed at high levels in Tet‐Off‐SDF‐1β BMSCs, augments the cell‐mediated therapeutic effects resulting in enhanced bone formation, as evidenced by ex vivo μCT and bone histomorphometry. The data demonstrate the specific contribution of SDF‐1β to BMSC‐mediated bone formation, and validate the feasibility of the Tet‐Off technology to regulate SDF‐1β expression in vivo. In conclusion, SDF‐1β provides potent synergistic effects supporting BMSC‐mediated bone formation and appears a suitable candidate for optimization of bone augmentation in translational protocols. Published 2014. This article has been contributed to by US Government employees and their work is in the public domain in the USA. J Orthop Res 33:174–184, 2015.

     

Prefabrication of a Vascularized Bone Graft With Beta Tricalcium Phosphate Using an In Vivo Bioreactor

We aimed to introduce an in vivo bioreactor‐vascular pedicle threaded through the central portion of a scaffold in which a vascularized bone graft was prefabricated using adenoviral human BMP‐2 gene (AdBMP2)‐modified bone marrow mesenchymal stem cells (BMSCs), beta tricalcium phosphate (β‐TCP), a vessel bundle, and muscularis membrane(group A). As controls, Adβgal‐BMSCs/β‐TCP granules, vessel bundle, and the muscularis membrane (group B); BMSCs/β‐TCP granules, vessel bundle, and muscularis membrane (group C); and β‐TCP granules, vessel bundle, and muscularis membrane (group D) were prepared. Formation of bone tissue and a vascular network was assessed by microangiography and histological methods 4 weeks after prefabrication. New cartilage and bone tissue in the space between β‐TCP granules (mainly endochondral bone) were confirmed by histology, and a de novo vascular network circulating from the vessel bundle through newly formed bone tissue was observed in group A. Formation of bone or cartilage was not observed in the control groups. We concluded that the in vivo bioreactor is a promising method for prefabrication of vascularized functional bone.     

Osteogenicity of autologous bone transplants in the goat

BACKGROUND: Little is known about the specific mechanisms that make autologous graft bone (AG) superior to the current alternatives. A potential mechanism is the active bone formation by the osteoprogenitor cells within the AG. However, whether these cells survive the transplantation is questionable, especially in nonvascularized, clinically sized grafts. In the present study, we investigated the role of viability in AG implanted ectopically and orthotopically in the goat. METHODS: Eight goats were operated on twice. At the first operation, pieces of vital or devitalized autologous cortical bone were implanted in the paraspinal muscles. Eight weeks later, corticocancellous plugs were taken from the femoral condyles, morselized, and reimplanted as either vital or devitalized orthotopic grafts. The goats received fluorochrome labels at 5, 7, and 9 weeks after the first operation. At 12 weeks, the goats were killed, and the samples were examined histologically. RESULTS: Ectopically, new bone had formed in both the vital and devitalized grafts. In the vital grafts, all three fluorochrome labels were present, indicating an early osteogenic mechanism. Within the devitalized grafts, only the 9-week label was observed. Histomorphometry indicated significantly more new bone in the vital grafts (10.3% vs. 1.7% in the devitalized grafts, P <0.01). Orthotopically, both vital and devitalized grafts showed new bone. Again, graft viability was advantageous in terms of new bone formation (14.5% vs. 9.3%, P <0.02). CONCLUSION: The cells inside the autologous bone transplants most likely survived transplantation and were capable of initiating and sustaining new bone formation.     

Current Application of β‐tricalcium Phosphate Composites in Orthopaedics

Presently, bioceramic materials have been extensively used in spinal surgery as bone grafts; however, there are some limitations for bioceramic materials. Calcium sulfate is rapidly absorbed in vivo, the degradation of which often occurs prior to the formation of new bones. Hydroxyapatite (HA) is hardly absorbed, which blocks the formation of new bones and remodeling, and results in poor local stability or permanent stress concentration. Only β‐tricalcium phosphate (β‐TCP) is relatively balanced between scaffold absorption and bone formation. And it is a good biodegradable ceramic material that could supply a large quantity of calcium ion and sulfate ion as well as scaffold structure for bone regeneration. However, the problem of single β‐TCP is lack of osteoinductivity and osteogenicity, which restricts its application. Therefore β‐TCP composite materials have been used in the field of orthopaedics in recent decades, which fully use excellent properties of other bone repairing materials, such as biodegradability, osteoinductivity, osteogenicity and osteoconductivity. These materials make up for the deficiencies of single β‐TCP and endow β‐TCP with more biological and physical properties.     

Fresh bone marrow introduction into porous scaffolds using a simple low‐pressure loading method for effective osteogenesis in a rabbit model

Recent advances in tissue engineering techniques have allowed porous biomaterials to be combined with osteogenic cells for effective bone regeneration. We developed a simple low‐pressure cell‐loading method using only syringes and stopcocks, and examined the effect of this method on osteogenesis when applied to the combination of highly porous β‐tricalcium phosphate (β‐TCP) and fresh autologous bone marrow. Both block and granule β‐TCP scaffolds were used to prepare implants in three different ways: without bone marrow as a control, with bone marrow that was allowed to penetrate spontaneously under atmospheric pressure (AP group), and with bone marrow that was seeded under low pressure (ULP group). These implants were transplanted into rabbit intramuscular sites, and the samples were examined biologically and histologically. The penetration efficiency of the block implants after marrow introduction was significantly higher in the ULP group than in the AP group. In the transplanted block samples, alkaline phosphatase activity was significantly higher in the ULP group at 2 weeks after implantation, and significantly more newly formed bone was observed in the ULP group at both 5 and 10 weeks compared with the AP group. Similar results were observed even in the experiment using β‐TCP granules, which are smaller than the blocks and frequently used clinically. Because of its convenience and safety, this low‐pressure method might be a novel, effective treatment to promote osteogenesis with bone marrow in clinical bone reconstruction surgeries. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:1–7, 2009     

组织工程骨修复牙槽嵴裂的实验研究

目的探讨骨髓来源的种子细胞复合胶原蛋白海绵构建组织工程骨修复牙槽嵴裂的可行性。方法12条实验犬被分成4组。于双侧上颌第3切牙处去除长15mm牙槽骨形成牙槽嵴裂动物模型;经股骨骨髓穿刺,分离骨髓基质干细胞,培养、传代扩增诱导后,与蛋白胶原海绵混合培养48h,植入骨缺损处。饲养12周后处死动物,通过三维CT及组织学检查评价骨缺损修复的效果。结果实验组牙槽骨断端间形成完整的骨连接,切片可见髓腔通畅,新形成的牙槽嵴宽度与松质骨组相似(P>0.05),但高度不足(P<0.05)。结论组织工程骨可以较好地修复牙槽嵴裂,有望成为修复牙槽嵴裂的治疗方法。     

Relevance of bone graft viability in a goat transverse process model

Little is known about the mechanism by which autologous bone grafts are so successful. The relevance of viable osteogenic cells, which is a prominent difference between autologous bone graft and conventional alternatives, is especially controversial. With the emergence of bone tissue engineering, knowledge of the exact role of these cells has become crucial. The most obvious question to answer is whether viability of the graft has an effect on bone formation. In the current study, we investigated this effect of bone graft viability in a transverse process model that represents the initial bone formation in posterolateral spinal fusion. Eight goats received viable and devitalized autologous bone grafts in chambers mounted on the decorticated lumbar transverse processes. In addition, five goats received empty chambers. Histology and histomorphometry were performed after a 12‐week implantation, and the dynamics of bone formation was monitored by sequential fluorochrome labeling. An obvious qualitative effect of viability was demonstrated by the presence of early onset osteogenesis distant from the transverse process bone in the viable grafts only. Quantitative analysis indicated about 30% more bone in the viable grafts, however, this difference was not statistically significant. In the empty chambers, bone was found in comparable quantities. We conclude that there is a qualitative advantage of graft viability in terms of early graft‐derived osteogenesis. However, this advantage did not lead to significantly more bone formation in the viable bone grafts. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 1055–1059, 2009     

Different bone remodeling levels of trabecular and cortical bone in response to changes in Wnt/β‐catenin signaling in mice

Trabecular bone and cortical bone have different bone remodeling levels, and the underlying mechanisms are not fully understood. In the present study, the expression of Wnt/β‐catenin signaling and its downstream molecules along with bone mass in trabecular and cortical bone were compared in wild‐type mice, constitutive activation of β‐catenin (CA‐β‐catenin) mice and β‐catenin deletion mice. It was found that the expression level of most of the examined genes such as Wnt3a, β‐catenin, osteocalcin and RANKL/OPG ratio were significantly higher in trabecular bone than in cortical bone in wild‐type mice. CA‐β‐catenin resulted in up‐regulated expression of the above‐mentioned genes except for RANKL/OPG ratio, which were down‐regulated. Also, CA‐β‐catenin led to increased number of osteoblasts, decreased number of osteoclasts and increased bone mass in both the trabecular bone and cortical bone compared with wild‐type mice; however, the extent of changes was much greater in the trabecular bone than in the cortical bone. By contrast, null β‐catenin led to down‐regulated expression of the above‐mentioned genes except for RANKL/OPG ratio. Furthermore, β‐catenin deletion led to decreased number of osteoblasts, increased number of osteoclasts and decreased bone mass when compared with wild‐type mice. Again, the extent of these changes was more significant in trabecular bone than cortical bone. Taken together, we found that the expression level of Wnt/β‐catenin signaling and bone remodeling‐related molecules were different in cortical bone and trabecular bone, and the trabecular bone was more readily affected by changes in the Wnt/β‐catenin signaling pathway. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:812–819, 2017.

     

骨髓间充质干细胞膜片复合磷酸三钙陶瓷构建工程化骨组织的体内成骨研究

目的：探讨骨髓间充质干细胞膜片复合磷酸三钙支架材料构建组织工程骨的可行性。方法：将兔骨髓间充质干细胞高密度接种于普通培养皿,在成骨诱导条件下连续培养2周,获得细胞膜片,修剪成长方形,并由一端卷起包裹圆柱状的磷酸三钙材料。静置孵育24h后将构建物移植到裸鼠背部皮下。术后6周取材,进行大体观察、组织学检查、组织定量学分析。结果：所获骨髓间充质干细胞膜片有多层细胞组成,保留了细胞外基质。实验组在材料表面及其孔隙内有较多的骨质形成;单一材料组空隙内为纤维组织,未见骨或软骨样组织;单一膜片组见片状编织骨形成。结论：骨髓间充质干细胞膜片在体内具有良好的成骨能力,可作为细胞释放载体与磷酸三钙支架复合构建骨组织。本研究为骨组织工程构建提供了新的方法。     

In vivo study on the healing of bone defects treated with bone marrow stromal cells, platelet-rich plasma, and freeze-dried bone allografts, alone and in combination.

The repair of confined trabecular bone defects in rabbits treated by autologous bone marrow stromal cells (BMSC), platelet-rich plasma (PRP), freeze-dried bone allografts (FDBA) alone and in combination (BMSC + PRP; FDBA + BMSC; FDBA + PRP; FDBA + PRP + BMSC) was compared. A critical size defect was created in the distal part of the femurs of 48 adult rabbits. Histology and histomorphometry were used in the evaluation of healing at 2, 4, and 12 weeks after surgery. The healing rate (%) was calculated by measuring the residual bone defect area. Architecture of the newly formed bone was compared with that of bone at the same distal femur area of healthy rabbits. The defect healing rate was higher in PRP + BMSC, FDBA + PRP, FDBA + BMSC, and FDBA + PRP + BMSC treatments, while lower values were achieved with PRP treatment at all experimental times. The highest bone-healing rate at 2 weeks was achieved with FDBA + PRP + BMSC treatment, which resulted significantly different from PRP (p < 0.05) and BMSC (p < 0.05) treatments. At 4 weeks, the bone-healing rate increased except for PRP treatment. Finally, the bone-healing rate of FDBA + PRP, FDBA + BMSC, and FDBA + PRP + BMSC was significantly higher than that of PRP at 12 weeks (p < 0.05). At 12 weeks, significant differences still existed between PRP, BMSC, and FDBA groups and normal bone (p < 0.05). These results showed that the combination of FDBA, BMSC and PRP permitted an acceleration in bone healing and bone remodeling processes.     

Differential analysis of peripheral blood‐ and bone marrow‐derived endothelial progenitor cells for enhanced vascularization in bone tissue engineering

For tissue engineering applications, effective bone regeneration requires rapid neo‐vascularization of implanted grafts to ensure the survival of cells in the early post‐implantation phase. Incorporation of autologous endothelial progenitor cells (EPCs) for the promotion of primitive vascular network formation ex vivo has offered great promise for improved graft survival, enhanced rate of vascularization and bone regeneration in vivo. For clinical usage, identification of an optimal EPC isolation source from the patient is critical. We have, for the first time, characterized and directly compared EPCs from rabbit peripheral blood and bone marrow (PB‐EPCs and BM‐EPCs, respectively). PB‐EPCs outperformed BM‐EPCs on all measures. PB‐EPCs displayed typical endothelial cell markers, such as CD31, as well as high angiogenic potential in three‐dimensional extracellular matrix in vitro. Furthermore, PB‐EPCs cultured simultaneously with mesenchymal stem cells, displayed significantly enhanced expression levels of key osteogenic and vascular markers, including alkaline phosphatase, bone morphogenetic protein 2, and vascular endothelial growth factor. On the contrary, putative BM‐EPCs did not express CD31, and instead, expressed key smooth muscle markers. BM‐EPCs further failed to display vasculogenic activity. Hence, the highly angiogenic PB‐derived EPCs may serve as an ideal cell population for enhanced vascularization and success of engineered bone tissue. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1507–1515, 2012     

Plasticizer di(2‐ethylhexyl)phthalate interferes with osteoblastogenesis and adipogenesis in a mouse model

Plasticizer di(2‐ethylhexyl)phthalate (DEHP) can leach from medical devices such as blood storage bags and the tubing. Recently, epidemiological studies showed that phthalate metabolites levels in the urine are associated with low bone mineral density (BMD) in older women. The detailed effect and mechanism of DEHP on osteoblastogenesis and adipogenesis, and bone loss remain to be clarified. Here, we investigated the effect and mechanism of DEHP and its active metabolite mono(2‐ethylhexyl)phthalate (MEHP) on osteoblastogenesis and adipogenesis. The in vitro study showed that osteoblast differentiation of bone marrow stromal cells (BMSCs) was significantly and dose‐dependently decreased by DEHP and MEHP (10–100 µM) without cytotoxicity to BMSCs. The mRNA expressions of alkaline phosphatase, Runx2, osteocalcin (OCN), Wnt1, and β‐catenin were significantly decreased in DEHP‐ and MEHP‐treated BMSCs during differentiation. MEHP, but not DEHP, significantly increased the adipocyte differentiation of BMSCs and PPARγ mRNA expression. Both DEHP and MEHP significantly increased the ratios of phosphorylated β‐catenin/β‐catenin and inhibited osteoblastogenesis, which could be reversed by Wnt activator lithium chloride and PPARγ inhibitor T0070907. Moreover, exposure of mice to DEHP (1, 10, and 100 mg/kg) for 8 weeks altered BMD and microstructure. In BMSCs isolated from DEHP‐treated mice, osteoblastogenesis and Runx2, Wnt1, and β‐catenin expression were decreased, but adipogenesis and PPARγ expression were increased. These findings suggest that DEHP and its metabolite MEHP exposure may inhibit osteoblastogenesis and promote adipogenesis of BMSCs through the Wnt/β‐catenin‐regulated and thus triggering bone loss. PPARγ signaling may play an important role in MEHP‐ and DEHP‐induced suppression of osteogenesis. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1124–1134, 2018.

     

μCT‐Based Measurement of Cortical Bone Graft‐to‐Host Union

Evaluation of structural bone grafts risk of failure requires noninvasive quantitative predictors of functional strength. We hypothesized that a quantitative graft‐to‐host union biometric would correlate significantly with biomechanical properties as a surrogate for the risk of fracture. To test this, we developed a novel algorithm to compute the union between host callus and graft, which was termed the union ratio. We compared the union ratio of live autografts to devitalized allografts implanted into the mid‐diaphysis of mouse femurs for 6 and 9 wk. Surprisingly, the autograft union ratio decreased from 0.228 ± 0.029 at 6 wk to 0.15 ± 0.011 at 9 wk (p < 0.05) and did not correlate with the torsional properties of the autografts. The allograft union ratio was 0.105 ± 0.023 at 6 wk but increased to 0.224 ± 0.029 at 9 wk (p < 0.05). As a single variable, the union ratio correlated significantly with ultimate torque (R² = 0.58) and torsional rigidity (R² = 0.51) of the allografts. Multivariable regression analyses of allografts that included the union ratio, the graft bone volume, the maximum and minimum polar moment of inertia, and their first‐order interaction terms with the union ratio as independent variables resulted in significant correlations with the ultimate torque and torsional rigidity (adjusted R² = 0.80 and 0.89, respectively). These results suggest that, unlike live autografts, the union between the devitalized allograft and host contributes significantly to the strength of grafted bone. The union ratio has important clinical implications as a novel biometric for noninvasive assessment of functional strength and failure risk.     

自体移植的大鼠骨髓间充质干细胞在脊髓损伤处的存活及分化

目的观察自体移植的骨髓间充质干细胞(BMSC)在脊髓损伤(SCI)处的存活及向神经细胞分化情况。方法将36只雄性SD大鼠随机分为PBS注射组(PBS组)和BMSC自体移植组(BMSC组)。BMSC组大鼠在术前均进行自体BMSC分离培养。2组大鼠均采用改良Allen撞击装置制备T9水平的SCI模型,PBS组于损伤处注射PBS,BMSC组注射第4代自体BMSC。于移植后2、3、5周采用Basso-Beattie-Bresnahan(BBB)评分法进行运动功能评分;移植后2、3、5周取损伤部位脊髓,采用Hoechst33342染色法观察移植细胞存活情况,同时行免疫荧光化学染色检测自体移植的BMSC中微管相关蛋白2(MAP-2)及胶质纤维酸性蛋白(GFAP)的表达情况。结果移植后2、3、5周,BMSC组大鼠BBB评分均高于PBS组,差异有统计学意义(P<0.05)。移植后2、3、5周,BMSC组大鼠损伤处脊髓有不同数量Hoechst33342标记细胞存活,其中2周时细胞存活较多,5周时细胞存活较少;移植后2、3周,未检测到移植细胞表达MAP-2及GFAP,5周时检测到部分移植细胞表达MAP-2和GFAP。结论SCI后立即进行移植的自体BMSC可在损伤处存活,部分存活细胞可向神经元和星形胶质细胞方向分化,促进大鼠后肢运动功能的恢复。     

Mechanical activation of β‐catenin regulates phenotype in adult murine marrow‐derived mesenchymal stem cells

Regulation of skeletal remodeling appears to influence the differentiation of multipotent mesenchymal stem cells (MSC) resident in the bone marrow. As murine marrow cultures are contaminated with hematopoietic cells, they are problematic for studying direct effects of mechanical input. Here we use a modified technique to isolate marrow‐derived MSC (mdMSC) from adult mice, yielding a population able to differentiate into adipogenic and osteogenic phenotypes that is devoid of hematopoietic cells. In pure mdMSC populations, a daily strain regimen inhibited adipogenic differentiation, suppressing expression of PPARγ and adiponectin. Strain increased β‐catenin and inhibition of adipogenesis required this effect. Under osteogenic conditions, strain activated β‐catenin signaling and increased expression of WISP1 and COX2. mdMSC were also generated from mice lacking caveolin‐1, a protein known to sequester β‐catenin: caveolin‐1^(?/?) mdMSC exhibited retarded differentiation along both adipogenic and osteogenic lineages but retained mechanical responses that involved β‐catenin activation. Interestingly, caveolin‐1^(?/?) mdMSC failed to express bone sialoprotein and did not form mineralized nodules. In summary, mdMSC from adult mice respond to both soluble factors and mechanical input, with mechanical activation of β‐catenin influencing phenotype. As such, these cells offer a useful model for studies of direct mechanical regulation of MSC differentiation and function. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1531–1538, 2010     

Enhanced healing of goat femur‐defect using BMP7 gene‐modified BMSCs and load‐bearing tissue‐engineered bone

Segmental defect regeneration is still a clinical challenge. In this study, we investigated the feasibility of bone marrow stromal cells (BMSCs) infected with adenoviral vector containing the bone morphogenetic protein 7 gene (AdBMP7) and load‐bearing to enhance bone regeneration in a critically sized femoral defect in the goat model. The defects were implanted with AdBMP7‐infected BMSCs/coral (BMP7 group) or noninfected BMSCs/coral (control group), respectively, stabilized with an internal fixation rod and interlocking nails. Bridging of the segmental defects was evaluated by radiographs monthly, and confirmed by biomechanical tests. Much callus was found in the BMP7 group, and nails were taken off after 3 months of implantation, indicating that regenerated bone in the defect can be remodeled by load‐bearing, whereas after 6 months in control group. After load‐bearing, it is about 5 months; the mechanical property of newly formed bone in the BMP7 group was restored, but 8 months in control group. Our data suggested that the BMP7 gene‐modified BMSCs and load‐bearing can promote bone regeneration in segmental defects. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:412–418, 2010     

β‐Arrestin2 Regulates RANKL and Ephrins Gene Expression in Response to Bone Remodeling in Mice

PTH‐stimulated intracellular signaling is regulated by the cytoplasmic adaptor molecule β‐arrestin. We reported that the response of cancellous bone to intermittent PTH is reduced in β‐arrestin2^?/? mice and suggested that β‐arrestins could influence the bone mineral balance by controlling RANKL and osteoprotegerin (OPG) gene expression. Here, we study the role of β‐arrestin2 on the in vitro development and activity of bone marrow (BM) osteoclasts (OCs) and Ephrins ligand (Efn), and receptor (Eph) mRNA levels in bone in response to PTH and the changes of bone microarchitecture in wildtype (WT) and β‐arrestin2^?/? mice in models of bone remodeling: a low calcium diet (LoCa) and ovariectomy (OVX). The number of PTH‐stimulated OCs was higher in BM cultures from β‐arrestin2^?/? compared with WT, because of a higher RANKL/OPG mRNA and protein ratio, without directly influencing osteoclast activity. In vivo, high PTH levels induced by LoCa led to greater changes in TRACP5b levels in β‐arrestin2^?/? compared with WT. LoCa caused a loss of BMD and bone microarchitecture, which was most prominent in β‐arrestin2^?/?. PTH downregulated Efn and Eph genes in β‐arrestin2^?/?, but not WT. After OVX, vertebral trabecular bone volume fraction and trabecular number were lower in β‐arrestin2^?/? compared with WT. Histomorphometry showed that OC number was higher in OVX‐β‐arrestin2^?/? compared with WT. These results indicate that β‐arrestin2 inhibits osteoclastogenesis in vitro, which resulted in decreased bone resorption in vivo by regulating RANKL/OPG production and ephrins mRNAs. As such, β‐arrestins should be considered an important mechanism for the control of bone remodeling in response to PTH and estrogen deprivation.     

Skeletal repair in rabbits using a novel biomimetic composite based on adipose‐derived stem cells encapsulated in collagen I gel with PLGA‐β‐TCP scaffold

In bone tissue engineering, the cell distribution mode in the scaffold may affect in vivo osteogenesis. Therefore, we fabricated a novel biomimetic construct based on a combination of rabbit adipose‐derived stem cells (rASCs) encapsulated in collagen I gel with a PLGA‐β‐TCP scaffold (rASCs‐COL/PLGA‐β‐TCP, group A), the combination of rASCs and PLGA‐β‐TCP (rASCs/PLGA‐β‐TCP, group B), the combination of collagen I gel and PLGA‐β‐TCP (COL/PLGA‐β‐TCP, group C), and PLGA‐β‐TCP scaffold (group D). The composites were implanted into a 15‐mm length critical‐sized segmental radial defect. The results were assessed by histology, radiographs, bone mineral density (BMD), and mechanical testing. After 24 weeks, the medullary cavity recanalized, bone was rebuilt, and molding finished, the bone contour remodeled smoothly and the scaffold degraded completely in group A. The BMDs and mechanical properties were similar to normal. However, the bone defect remained unrepaired in groups B, C, and D. Moreover, the scaffold degradation rate in group A was significantly higher than the other groups. Thus, enhanced in vivo osteogenesis of rASCs wrapped in collagen I gel combined with PLGA‐β‐TCP was achieved, and the bone defect was repaired. We hope this study provides new insights into ASCs‐based bone tissue engineering. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:252–257, 2010     

Autologous bone‐marrow‐derived mesenchymal stem cell transplantation into injured rat urethral sphincter

Objectives: To evaluate the functional and histological recovery by autologous bone‐marrow‐derived mesenchymal stem cell (BMSC) transplantation into injured rat urethral sphincters. Methods: BMSC were harvested from female Sprague–Dawley retired breeder rats for later transplantation. The cells were cultured, and transfected with the green fluorescence protein gene. The urethral sphincters were injured by combined urethrolysis and cardiotoxin injection. One week after injury, the cultured BMSC were injected autologously into the periurethral tissues. Controls included sham‐operated rats and injured rats injected with cell‐free medium (CFM). Abdominal leak point pressures (LPP) were measured before and after surgery during the following 13 weeks. The urethras were then retrieved for histological evaluation. The presence of green‐fluorescence‐protein‐labeled cells and the regeneration of skeletal muscles, smooth muscles, and peripheral nerves were evaluated by immunohistochemical staining. Results: LPP was significantly reduced in the injured rats. It increased gradually after transplantation, but there was no significant difference between the BMSC and CFM groups. In the BMSC group, transplanted cells survived and differentiated into striated muscle cells and peripheral nerve cells. The proportions of skeletal muscle cells and peripheral nerves in the urethra were significantly greater in the BMSC group compared to the CFM group. Conclusions: Despite a clear trend towards recovery of LPP in BMSC‐transplanted urethras, no significant effect was detected. Further study is required for clinical applications for the treatment of stress urinary incontinence.     

Isolation and characterization of turkey bone marrow‐derived mesenchymal stem cells

Flexor tendon injury is often associated with suboptimal outcomes and results in substantial digit dysfunction. Stem cells have been isolated from several experimental animals for the growing interest and needs of utilizing cell‐based therapies. Recently, turkey has been developed as a new large animal model for flexor tendon research. In the present study, we reported the isolation and characterization of bone marrow‐derived mesenchymal stem cells (BMSCs) from 8‐ to 12‐month‐old heritage‐breed turkeys. The isolated cells demonstrated fibroblast‐like morphology, clonogenic capacity, and high proliferation rate. These cells were positive for surface antigens CD90, CD105, and CD44, but were negative for CD45. The multipotency of turkey BMSCs was determined by differentiating cells into osteogenic, adipogenic, chondrogenic, and tenogenic lineages. There was upregulated gene expression of tenogenic markers, including mohawk, tenomodulin, and EGR1 as well as increased collagen synthesis in BMP12 induced cells. The successful isolation and verification of bone marrow‐derived MSCs from turkey would provide opportunities of studying cell‐based therapies and developing new treatments for tendon injuries using this novel preclinical large animal model. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1419–1428, 2019.