In vivo bone formation following transplantation of human adipose-derived stromal cells that are not differentiated osteogenically

A number of studies have shown in vivo bone regeneration by transplantation of osteogenic cells differentiated in vitro from adipose-derived stromal cells (ADSCs). However, the in vitro osteogenic differentiation process requires an additional culture period, and the dexamethasone that is generally used in the process may be cytotoxic. Here, we tested the hypothesis that ADSCs that are not differentiated osteogenically in vitro prior to transplantation would extensively regenerate bone in vivo when exogenous bone morphogenetic protein-2 (BMP-2) is delivered to the transplantation site. We fabricated a poly(dl-lactic-co-glycolic acid)/hydroxyapatite (PLGA/HA) composite scaffold with osteoactive HA that is highly exposed on the scaffold surface. This scaffold was able to release BMP-2 over a 4-week period in vitro. Human ADSCs cultured on BMP-2-loaded PLGA/HA scaffolds for 2 weeks differentiated toward osteogenic cells expressing alkaline phosphatase (ALP), osteopontin (OPN), and osteocalcin (OCN) mRNA, while cells on PLGA/HA scaffolds without BMP-2 expressed only ALP. To study in vivo bone formation, PLGA/HA scaffolds (group 1), BMP-2-loaded PLGA/HA scaffolds (group 2), undifferentiated ADSCs seeded on PLGA/HA scaffolds (group 3), and undifferentiated ADSCs seeded on BMP-2-loaded PLGA/HA scaffolds (group 4) were implanted into dorsal, subcutaneous spaces of athymic mice. Eight weeks after implantation, group 4 exhibited a 25-fold greater bone formation area and 5-fold higher calcium deposition than group 3. Bone regeneration by transplanted human ADSCs in group 4 was confirmed by expression of human-specific osteoblastic genes, ALP, collagen type I, OPN, OCN, and bone sialoprotein, while group 3 expressed much lower levels of collagen type I and OPN mRNA only. This study demonstrates the feasibility of extensive in vivo bone regeneration by transplantation of ADSCs without prior in vitro osteogenic differentiation, and that a PLGA/HA composite BMP-2 delivery system stimulates bone regeneration following transplantation of undifferentiated human ADSCs.     

Engineering bone-like tissue in vitro using human bone marrow stem cells and silk scaffolds

Porous biodegradable silk scaffolds and human bone marrow derived mesenchymal stem cells (hMSCs) were used to engineer bone-like tissue in vitro. Two different scaffolds with the same microstructure were studied: collagen (to assess the effects of fast degradation) and silk with covalently bound RGD sequences (to assess the effects of enhanced cell attachment and slow degradation). The hMSCs were isolated, expanded in culture, characterized with respect to the expression of surface markers and ability for chondrogenic and osteogenic differentiation, seeded on scaffolds, and cultured for up to 4 weeks. Histological analysis and microcomputer tomography showed the development of up to 1.2-mm-long interconnected and organized bonelike trabeculae with cuboid cells on the silk-RGD scaffolds, features still present but to a lesser extent on silk scaffolds and absent on the collagen scaffolds. The X-ray diffraction pattern of the deposited bone corresponded to hydroxyapatite present in the native bone. Biochemical analysis showed increased mineralization on silk-RGD scaffolds compared with either silk or collagen scaffolds after 4 weeks. Expression of bone sialoprotein, osteopontin, and bone morphogenetic protein 2 was significantly higher for hMSCs cultured in osteogenic than control medium both after 2 and 4 weeks in culture. The results suggest that RGD-silk scaffolds are particularly suitable for autologous bone tissue engineering, presumably because of their stable macroporous structure, tailorable mechanical properties matching those of native bone, and slow degradation.     

Development of silk-based scaffolds for tissue engineering of bone from human adipose-derived stem cells

Silk fibroin is a potent alternative to other biodegradable biopolymers for bone tissue engineering (TE), because of its tunable architecture and mechanical properties, and its demonstrated ability to support bone formation both in vitro and in vivo. In this study, we investigated a range of silk scaffolds for bone TE using human adipose-derived stem cells (hASCs), an attractive cell source for engineering autologous bone grafts. Our goal was to understand the effects of scaffold architecture and biomechanics and use this information to optimize silk scaffolds for bone TE applications. Silk scaffolds were fabricated using different solvents (aqueous vs. hexafluoro-2-propanol (HFIP)), pore sizes (250-500 μm vs. 500-1000 μm) and structures (lamellar vs. spherical pores). Four types of silk scaffolds combining the properties of interest were systematically compared with respect to bone tissue outcomes, with decellularized trabecular bone (DCB) included as a "gold standard". The scaffolds were seeded with hASCs and cultured for 7 weeks in osteogenic medium. Bone formation was evaluated by cell proliferation and differentiation, matrix production, calcification and mechanical properties. We observed that 400-600 μm porous HFIP-derived silk fibroin scaffold demonstrated the best bone tissue formation outcomes, as evidenced by increased bone protein production (osteopontin, collagen type I, bone sialoprotein), enhanced calcium deposition and total bone volume. On a direct comparison basis, alkaline phosphatase activity (AP) at week 2 and new calcium deposition at week 7 were comparable to the cells cultured in DCB. Yet, among the aqueous-based structures, the lamellar architecture induced increased AP activity and demonstrated higher equilibrium modulus than the spherical-pore scaffolds. Based on the collected data, we propose a conceptual model describing the effects of silk scaffold design on bone tissue formation.     

Initial cell pre-cultivation can maximize ECM mineralization by human mesenchymal stem cells on silk fibroin scaffolds

Fast remineralization of bone defects by means of tissue engineering is one of many targets in orthopedic regeneration. This study investigated the influence of a range of pre-culture durations for human bone marrow derived mesenchymal stem cells (hMSC) before inducing differentiation into osteoblast-like cells. The aim was to find the conditions that lead to maximal extracellular matrix (ECM) mineralization, in terms of both amount and best distribution. Additionally, the influence of silk fibroin scaffold pore size on mineralization was assessed. The formation of mineralized ECM by hMSCs cultured in osteogenic medium on silk fibroin scaffolds was monitored and quantified for up to 72 days in culture using non-invasive time-lapse micro-computed tomography (micro-CT). ECM mineralization increased linearly 3 weeks after the beginning of the experiment with addition of differentiation medium. Biochemical end-point assays measured the amount of DNA, calcium deposits, alkaline phosphatase activity and cell metabolic activity to corroborate the hypothesis that an initial pre-culture period of hMSCs on silk fibroin scaffolds can accelerate mineralized ECM formation. According to the micro-CT analysis mineralization on silk fibroin scaffolds with pores of 112-224 μm diameter was most efficient with an initial cell pre-culture period of 9 days, showing 6.87±0.81× higher mineralization values during the whole cultivation period than without an initial cell pre-culture period.     

Bone morphogenetic protein 2 and retinoic acid accelerate in vivo bone formation, osteoclast recruitment, and bone turnover

Reconstruction of craniofacial defects presents a substantial biomedical burden, and requires complex surgery. Interestingly, children after age 2 years and adults are unable to heal large skull defects. This nonhealing paradigm provides an excellent model system for craniofacial skeletal tissueengineering strategies. Previous studies have documented the in vivo osteogenic potential of adipose-derived stromal (ADS) cells and bone marrow-derived stromal (BMS) cells. This study investigates the ability to accelerate in vivo osteogenesis on ex vivo recombinant human bone morphogenetic protein 2 (BMP-2) and retinoic acid stimulation. Mouse osteoblasts, ADS cells, and BMS cells were seeded onto apatite-coated PLGA scaffolds, stimulated with rhBMP-2 and retinoic acid ex vivo for 4 weeks, and subsequently implanted into critically sized (4 mm) calvarial defects. Samples were harvested after 2, 4, 8, and 12 weeks. Areas of complete bony bridging were noted as early as 2 weeks in vivo; however, osteoclasts were attracted to the scaffold as identified by calcitonin receptor staining and tartrate-resistant acid phosphatase activity staining. Although the optimal method of in vitro osteogenic priming for mesenchymal cells remains unknown, these results provide evidence that BMP-2 and retinoic acid stimulation of multipotent cells ex vivo can subsequently induce significant quantities of bone formation within a short time period in vivo.     

Osteogenesis by human mesenchymal stem cells cultured on silk biomaterials: comparison of adenovirus mediated gene transfer and protein delivery of BMP-2

Bone tissue engineering, gene therapy based on human mesenchymal stem cells (MSCs) and silk fibroin biomaterials were combined to study the impact of viral transfection on MSC osteogenic performance in vitro. MSCs were transduced with adenovirus containing a human BMP-2 (Ad-BMP-2) gene at clinically reasonable viral concentrations and cultured for 4 weeks. Controls with nontransfected MSCs, but exposed to exogenous BMP-2 concentrations on an analogous time profile as that secreted by the Ad-BMP-2 group, were compared. Both the Ad-BMP-2 MSC group and the exogenous protein BMP-2 group strongly expressed osteopontin and bone sialoprotein. Cells secreted a matrix that underwent mineralization on the silk fibroin scaffolds, forming clusters of osseous material, as determined by micro-computed tomography. The expression of osteogenic marker proteins and alkaline phosphatase was significantly higher in the Ad-BMP-2 MSC group than in the exogenous protein BMP-2 group, and no significant differences in mineralization were observed in two of the three MSC sources tested. The results demonstrate that transfection resulted in higher levels of expression of osteogenic marker genes, no change in proliferation rate and did not impact the capacity of the cells to calcify tissues on these protein scaffolds. These findings suggest additional options to control differentiation where exogenous additions of growth factors or morphogens can be replaced with transfected MSCs.     

Bioactive cell-derived matrices combined with polymer mesh scaffold for osteogenesis and bone healing

Successful bone tissue engineering generally requires an osteoconductive scaffold that consists of extracellular matrix (ECM) to mimic the natural environment. In this study, we developed a PLGA/PLA-based mesh scaffold coated with cell-derived extracellular matrix (CDM) for the delivery of bone morphogenic protein (BMP-2), and assessed the capacity of this system to provide an osteogenic microenvironment. Decellularized ECM from human lung fibroblasts (hFDM) was coated onto the surface of the polymer mesh scaffolds, upon which heparin was then conjugated onto hFDM via EDC chemistry. BMP-2 was subsequently immobilized onto the mesh scaffolds via heparin, and released at a controlled rate. Human placenta-derived mesenchymal stem cells (hPMSCs) were cultured in such scaffolds and subjected to osteogenic differentiation for 28 days in vitro. The results showed that alkaline phosphatase (ALP) activity, mineralization, and osteogenic marker expression were significantly improved with hPMSCs cultured in the hFDM-coated mesh scaffolds compared to the control and fibronectin-coated ones. In addition, a mouse ectopic and rat calvarial bone defect model was used to examine the feasibility of current platform to induce osteogenesis as well as bone regeneration. All hFDM-coated mesh groups exhibited a significant increase of newly formed bone and in particular, hFDM-coated mesh scaffold loaded with a high dose of BMP-2 exhibited a nearly complete bone defect healing as confirmed via micro-CT and histological observation. This work proposes a great potency of using hFDM (biophysical) coupled with BMP-2 (biochemical) as a promising osteogenic microenvironment for bone tissue engineering applications.     

Biocompatibility and osteogenesis of biomimetic Bioglass-Collagen-Phosphatidylserine composite scaffolds for bone tissue engineering

A novel biomimetic composite scaffold Bioglass-Collagen-Phosphatidylserine (BG-COL-PS) was fabricated with a freeze-drying technique. The macrostructure and morphology as well as mechanical strength of the scaffolds were characterized. Scanning electronic microscopy (SEM) showed that the BG-COL-PS scaffolds exhibited interconnected porous structures with pore sizes of several microns up to about 300 μm. The scaffolds have a porosity of 75.40% and the corresponding compressive strength of 1.5469 Mpa. Rat mesenchymal stem cells (rMSCs) were seeded on BG-COL-PS or BG-COL scaffolds and cultured for 21 days in vitro. Based on the results of SEM, dsDNA content, alkaline phosphatase (ALP) activity, osteogenic gene expression analysis and alizarin red staining, the responses of MSCs to the scaffold exhibited a higher degree of attachment, growth as well as osteogenic differentiation than those on BG-COL scaffolds in vitro. To investigate the in vivo biocompatibility and osteogenesis of the composite scaffolds, both pure BG-COL-PS scaffolds and MSC/scaffold constructs were implanted in rat femurs defects for 6 weeks and studied histologically and radiographically. The in vivo results showed that BG-COL-PS composite scaffolds exhibited good biocompatibility and extensive osteoconductivity with host bone. Moreover, the BG-COL-PS/MSC constructs dramatically enhanced the efficiency of new bone formation than pure BG-COL-PS scaffolds or BG-COL/MSC constructs. All these results demonstrate the usefulness of PS composited BG-COL-PS scaffolds for inducing enhanced bone formation. The BG-COL-PS scaffolds fulfill the basic requirements of bone tissue engineering scaffold and have the potential to be applied in orthopedic and reconstructive surgery.     

Nucleation and growth of mineralized bone matrix on silk-hydroxyapatite composite scaffolds

We describe a composite hydroxyapatite (HA)-silk fibroin scaffold designed to induce and support the formation of mineralized bone matrix by human mesenchymal stem cells (hMSCs) in the absence of osteogenic growth factors. Porous three-dimensional silk scaffolds were extensively used in our previous work for bone tissue engineering and showed excellent biodegradability and biocompatibility. However, silk is not an osteogenic material and has a compressive stiffness significantly lower than that of native bone. In the present study, we explored the incorporation of silk sponge matrices with HA (bone mineral) micro-particles to generate highly osteogenic composite scaffolds capable of inducing the in vitro formation of tissue-engineered bone. Different amounts of HA were embedded in silk sponges at volume fractions of 0%, 1.6%, 3.1% and 4.6% to enhance the osteoconductive activity and mechanical properties of the scaffolds. The cultivation of hMSCs in the silk/HA composite scaffolds under perfusion conditions resulted in the formation of bone-like structures and an increase in the equilibrium Young's modulus (up to 4-fold or 8-fold over 5 or 10 weeks of cultivation, respectively) in a manner that correlated with the initial HA content. The enhancement in mechanical properties was associated with the development of the structural connectivity of engineered bone matrix. Collectively, the data suggest two mechanisms by which the incorporated HA enhanced the formation of tissue engineered bone: through osteoconductivity of the material leading to increased bone matrix production, and by providing nucleation sites for new mineral resulting in the connectivity of trabecular-like architecture.     

Osteogenesis on nanoparticulate mineralized collagen scaffolds via autogenous activation of the canonical BMP receptor signaling pathway

Skeletal regenerative medicine frequently incorporates deliverable growth factors to stimulate osteogenesis. However, the cost and side effects secondary to supraphysiologic dosages of growth factors warrant investigation of alternative methods of stimulating osteogenesis for clinical utilization. In this work, we describe growth factor independent osteogenic induction of human mesenchymal stem cells (hMSCs) on a novel nanoparticulate mineralized collagen glycosaminoglycan scaffold (MC-GAG). hMSCs demonstrated elevated osteogenic gene expression and mineralization on MC-GAG with minimal to no effect upon addition of BMP-2 when compared to non-mineralized scaffolds (Col-GAG). To investigate the intracellular pathways responsible for the increase in osteogenesis, we examined the canonical and non-canonical pathways downstream from BMP receptor activation. Constitutive Smad1/5 phosphorylation with nuclear translocation occurred on MC-GAG independent of BMP-2, whereas Smad1/5 phosphorylation depended on BMP-2 stimulation on Col-GAG. When non-canonical BMPR signaling molecules were examined, ERK1/2 phosphorylation was found to be decreased in MC-GAG but elevated in Col-GAG. No differences in Smad2/3 or p38 activation were detected. Collectively, these results demonstrated that MC-GAG scaffolds induce osteogenesis without exogenous BMP-2 addition via endogenous activation of the canonical BMP receptor signaling pathway.     

In vivo bone formation from human embryonic stem cell-derived osteogenic cells in poly(d,l-lactic-co-glycolic acid)/hydroxyapatite composite scaffolds

We have previously reported the efficient osteogenic differentiation of human embryonic stem cells (hESCs) by co-culture with primary human bone-derived cells (hPBDs) without the use of exogenous factors. In the present study, we explored whether osteogenic cells derived from hESCs (OC-hESCs) using the previously reported method would be capable of regenerating bone tissue in vivo. A three-dimensional porous poly(d,l-lactic-co-glycolic acid)/hydroxyapatite composite scaffold was used as a cell delivery vehicle. In vivo implantation of OC-hESC-seeded scaffolds showed significant bone formation in the subcutaneous sites of immunodeficient mice at 4 and 8 weeks after implantation (n=5 for each time point). Meanwhile, implantation of the control no cell-seeded scaffolds or human dermal fibroblast-seeded scaffolds did not show any new bone formation. In addition, the presence of BMP-2 (1 microg/scaffold) enhanced new bone tissue formation in terms of mineralization and the expression of bone-specific genetic markers. According to FISH analysis, implanted OC-hESCs remained in the regeneration sites, which suggested that the implanted cells participated in the formation of new bone. In conclusion, OC-hESCs successfully regenerated bone tissue upon in vivo implantation, and this regeneration can be further enhanced by the administration of BMP-2. These results suggest the clinical feasibility of OC-hESCs as a good alternative source of cells for bone regeneration.     

Enhancement of osteogenesis by poly(lactide-co-glycolide) sponges loaded with surface-embedded hydroxyapatite particles and rhBMP-2

The bone mesenchymal stem cells (BMSCs) were seeded on [poly(lactide-co-glycolide) scaffolds with hydroxyapatite (HA) coating, and "s" stands for surface] (PLGA/HA-S), PLGA/HA-M (containing the same HA amount in the matrix as that of the PLGA/HA-S and "m" stands for matrix), and PLGA scaffolds, which were then cultured in a medium-containing Escherichia coli-derived recombinant human bone morphogenetic protein-2 (ErhBMP-2). In vitro culture of rat BMSCs found no different cell morphology in all the scaffolds, but the alkaline phosphatase activity and osteogenic gene expression of type I collagen (COL I) and osteocalcin (OCN) in the PLGA/HA-S scaffolds were always highest and were significantly improved in comparison with those in the PLGA scaffolds. In a rat calvarial defect model, new bone formation was enhanced in the PLGA/HA-S/ErhBMP-2 implants at 4 and 8 weeks after implantation too. Therefore, the PLGA/HA-S scaffold can better enhance the ErhBMP-2-induced osteogenic differentiation of BMSCs in vitro and osteogenesis in vivo.     

复合骨碎补总黄酮的丝素蛋白/壳聚糖支架在兔关节软骨损伤中的应用研究

目的 以丝素蛋白/壳聚糖支架为载体将骨碎补总黄酮应用于兔软骨损伤局部,观察修复效果,为临床提供实验数据。方法 制备丝素蛋白/壳聚糖支架、骨碎补总黄酮缓释微球与负载骨碎补总黄酮缓释微球的丝素蛋白/壳聚糖支架,扫描电子显微镜下观察支架形貌,同时检测该支架的体外缓释能力。24只新西兰大白兔随机分3组,利用电钻在股骨滑车部位构建直径3.5 mm、深1.5 mm的软骨损伤模型,空白组软骨缺损处不植入任何材料,对照组植入单纯的丝素蛋白/壳聚糖支架,实验组植入负载骨碎补总黄酮缓释微球的丝素蛋白/壳聚糖支架,术后12周、24周行标本大体与组织学观察,RT-PCR检测修复组织Sox-9、II型胶原与聚集蛋白聚糖mRNA的表达量,Western blot检测软骨缺损部位II型胶原蛋白表达,分析软骨修复效果。结果 丝素蛋白/壳聚糖支架具有良好的三维孔隙结构,孔洞之间相互联通;制备的载药微球表面较光滑,为较规则的圆球形;载药微球均匀分散于丝素蛋白/壳聚糖支架基质中。丝素蛋白/壳聚糖支架可在体外持续稳定地释放骨碎补总黄酮,实验组软骨损伤修复效果优于对照组,对应的ICRS评分与Wakitani组织学评分高于对照组...     

Mandibular repair in rats with premineralized silk scaffolds and BMP-2-modified bMSCs

Premineralized silk fibroin protein scaffolds (mSS) were prepared to combine the osteoconductive properties of biological apatite with aqueous-derived silk scaffold (SS) as a composite scaffold for bone regeneration. The aim of present study was to evaluate the effect of premineralized silk scaffolds combined with bone morphogenetic protein-2 (BMP-2) modified bone marrow stromal cells (bMSCs) to repair mandibular bony defects in a rat model. bMSCs were expanded and transduced with adenovirus AdBMP-2, AdLacZ gene in vitro. These genetically modified bMSCs were then combined with premineralized silk scaffolds to form tissue-engineered bone. Mandibular repairs with AdBMP-2 transduced bMSCs/mSS constructs were compared with those treated with AdLacZ-transduced bMSCs/mSS constructs, native (nontransduced) bMSCs/mSS constructs and mSS alone. Eight weeks after post-operation, the mandibles were explanted and evaluated by radiographic observation, micro-CT, histological analysis and immunohistochemistry. The presence of BMP-2 gene enhanced tissue-engineered bone in terms of the most new bone formed and the highest local bone mineral densities (BMD) found. These results demonstrated that premineralized silk scaffold could serve as a potential substrate for bMSCs to construct tissue-engineered bone for mandibular bony defects. BMP-2 gene therapy and tissue engineering techniques could be used in mandibular repair and bone regeneration.