Maxillary sinus floor elevation using a tissue-engineered bone with rhBMP-2-loaded porous calcium phosphate cement scaffold and bone marrow stromal cells in rabbits

The aim of this study was to evaluate the effects of maxillary sinus floor elevation by a tissue-engineered bone complex with recombinant human bone morphogenetic protein-2 (rhBMP-2)-loaded porous calcium phosphate cement (CPC) scaffold and bone marrow stromal cells (bMSCs) in rabbits. bMSCs were cultured and osteogenically induced. The osteoblastic differentiation of expanded bMSCs was detected by alkaline phosphatase activity, and calcium deposits in vitro. Thirty-six rabbits were randomly allocated into week 2, 4 and 8 observation groups. At each time point, 24 maxillary sinus floor elevation surgeries in 12 rabbits were performed bilaterally and randomly implanted by (1) CPC materials alone (group A, n = 6), (2) rhBMP-2/CPC composite materials alone (group B, n = 6), (3) CPC/bMSCs complex (group C, n = 6) and (4) rhBMP-2/CPC/bMSCs complex (group D, n = 6). As for maxillary sinus floor elevation, rhBMP-2-loaded CPC could promote new bone formation as compared to CPC, while addition of bMSCs could further enhance its new bone formation and maturity significantly, as detected by histological findings, and fluorochrome labeling. Our data suggested that rhBMP-2/CPC possessed excellent osteoinductive ability, while combining with bMSCs could further promote new bone formation and maturation in maxillary sinus elevation.     

Engineering of bone using porous calcium phosphate cement and bone marrow stromal cells for maxillary sinus augmentation with simultaneous implant placement in goats

The aim of this study was to explore the effects of maxillary sinus floor elevation and simultaneous dental implantation with a tissue-engineered bone complex of calcium phosphate cement (CPC) scaffolds combined with bone marrow stromal cells (BMSCs). A large animal goat model is used with the tissue engineering method. Eighteen bilateral maxillary sinus of nine goats were randomly allocated into three groups; the CPC/BMSC complex (n=6) was used to elevate maxillary sinus floor with a simultaneous implant placement; the effects were compared with those treated with CPC alone (n=6) or autogenous bone (n=6). After a healing period of 3 months, sequential triad-color fluorescence labeling, micro-CT, as well as histological and histomorphometric analyses indicated that the tissue-engineered BMSC/CPC complex could promote earlier bone formation and mineralization, and maximally maintain the volume and height of the augmented maxillary sinus. By comparison, CPC-alone or autogenous bone achieved less bone formation and later mineralization. Besides, the average bone-implant contact value reflecting the osseointegration was 35.63%±9.42% in the BMSCs/CPC group, significantly higher than 22.47%±4.28% in the CPC-alone group or 28.26%±8.03% in the autogenous bone group. In conclusion, CPC serves as a potential substrate for BMSCs for the maxillary sinus floor augmentation and simultaneous implantation. The tissue-engineered bone might enhance the stability of implants and thus be of great significance to achieve improved quality to restore the oral function in clinic.     

Self-setting bioactive calcium-magnesium phosphate cement with high strength and degradability for bone regeneration

Calcium phosphate cement (CPC) has been successfully used in clinics as bone repair biomaterial for many years. However, poor mechanical properties and a low biodegradation rate limit any further applications. Magnesium phosphate cement (MPC) is characterized by fast setting, high initial strength and relatively rapid degradation in vivo. In this study, MPC was combined with CPC to develop novel calcium-magnesium phosphate cement (CMPC). The setting time, compressive strength, phase composition of hardened cement, degradation in vitro, cells responses in vitro by MG-63 cell culture and tissue responses in vivo by implantation of CMPC in bone defect of rabbits were investigated. The results show that CMPC has a shorter setting time and markedly better mechanical properties than either CPC or MPC. Moreover, CMPC showed significantly improved degradability compared to CPC in simulated body fluid. Cell culture results indicate that CMPC is biocompatible and could support cell attachment and proliferation. To investigate the in vivo biocompatibility and osteogenesis, the CMPC samples were implanted into bone defects in rabbits. Histological evaluation showed that the introduction of MPC into CPC enhanced the efficiency of new bone formation. CMPC also exhibited good biocompatibility, biodegradability and osteoconductivity with host bone in vivo. The results obtained suggest that CMPC, having met the basic requirements of bone tissue engineering, might have a significant clinical advantage over CPC, and may have the potential to be applied in orthopedic, reconstructive and maxillofacial surgery.     

Long-term outcome of cryopreserved bone-derived osteoblasts for bone regeneration in vivo

Cryopreserved bone-derived osteoblasts (CBOs) have been considered as a promising cell source for bone regeneration. Previous studies have demonstrated that CBOs had good proliferation and osteogenicity. However, the long-term outcome of CBOs in vivo still remains unknown. In this experiment, we applied CBOs combined with calcium phosphate cement (CPC) to augment maxillary sinus in canine, computer tomography, polychrome labeling, biomechanical tests, fluorescent immunohistochemistry staining and histological analysis were used to analyze the property and mineralization process of the tissue-engineered bone preclinical application. Our results showed that CBOs combined with CPC could promote bone regeneration, dramatically maintain the height, volume and biomechanical property of augmented maxillary sinus. Furthermore, the tissue-engineered bone was more mature than scaffold alone or autogenous bone, and bone formation and remodeling were still apparent 20 months postoperatively. Additionally, 4 months after surgery might be the suitable time point for implants placement in the regenerated bone. These results also indicate that cryopreserved bone may be a potential source of osteoblasts for maxillary sinus augmentation.     

Magnesium modification of a calcium phosphate cement alters bone marrow stromal cell behavior via an integrin-mediated mechanism

The chemical composition, structure and surface characteristics of biomaterials/scaffold can affect the adsorption of proteins, and this in turn influences the subsequent cellular response and tissue regeneration. With magnesium/calcium phosphate cements (MCPC) as model, the effects of magnesium (Mg) on the initial adhesion and osteogenic differentiation of bone marrow stromal cells (BMSCs) as well as the underlying mechanism were investigated. A series of MCPCs with different magnesium phosphate cement (MPC) content (0∼20%) in calcium phosphate cement (CPC) were synthesized. MCPCs with moderate proportion of MPC (5% and 10%, referred to as 5MCPC and 10MCPC) were found to effectively modulate the orientation of the adsorbed fibronectin (Fn) to exhibit enhanced receptor binding affinity, and to up-regulate integrin α5β1 expression of BMSCs, especially for 5MCPC. As a result, the attachment, morphology, focal adhesion formation, actin filaments assembly and osteogenic differentiation of BMSCs on 5MCPC were strongly enhanced. Further in vivo experiments confirmed that 5MCPC induced promoted osteogenesis in comparison to ot her CPC/MCPCs. Our results also suggested that the Mg on the underlying substrates but not the dissolved Mg ions was the main contributor to the above positive effects. Based on these results, it can be inferred that the specific interaction of Fn and integrin α5β1 had predominant effect on the MCPC-induced enhanced cellular response of BMSCs. These results provide a new strategy to regulate BMSCs adhesion and osteogenic differentiation by adjusting the Mg/Ca content and distribution in CPC, guiding the development of osteoinductive scaffolds for bone tissue regeneration.     

脱基质小牛骨粉复合骨髓基质干细胞的成骨作用

背景：利用骨组织工程技术在上颌窦提升中的成骨研究是目前口腔种植学到研究热点。 目的：探讨骨组织工程在上颌窦底提升同期牙种植中的成骨效果。 方法：体外分离培养犬骨髓基质干细胞,将细胞与脱基质小牛骨粉复合培养并向成骨细胞定向诱导分化。健康成年犬12只行双侧上颌窦底提升同期牙种植,一侧植入复合物,另一侧植入脱基质小牛骨粉做对照。 结果与结论：种植体稳固无松动,上颌窦黏膜完整。实验侧可见新生骨形成较早、骨量较多;对照侧新生骨形成较慢。X射线显示实验侧新生骨质致密,与种植体结合紧密。随时间的增加,牵出力增大,12,24周时两侧差异有显著性意义(P < 0.05)。组织形态学检测显示新生骨面积逐渐增加,12,24周时差异有显著性意义(P < 0.05)。提示组织工程化骨在上颌窦提升同期牙种植中可获得良好的成骨效果。     

新型组织工程化骨材料植入动物体内的成血管效应

背景：以生长因子、种子细胞、载体支架为基础的骨组织工程研究取得的成功,向人们展示了再造骨组织器官的美好前景,然而在临床应用方面往往效果不理想。其中很重要一个原因是组织工程骨很大程度上受制于移植物血管网缺乏造成的细胞供养障碍而导致失效。 目的：新型组织工程骨修复材料植入新西兰兔桡骨缺损处观察其成血管作用。 方法：将聚乳酸-聚羟基乙酸共聚物包裹碱性成纤维细胞生长因子制备成微球囊,然后与磷酸钙骨水泥混合,并与体外培养的同种异体骨髓间充质干细胞共培养制备新型组织工程骨修复材料。60只成年新西兰兔建立15 mm桡骨缺损模型后随机分成2组,实验组植入新型组织工程骨修复材料,对照组植入复合骨髓间充质干细胞的聚乳酸-聚羟基乙酸共聚物与磷酸钙骨水泥的混合材料。于术后4、8、12周,通过组织细胞形态学观察、核素骨扫描等手段,观察各个时期血管形成情况。 结果与结论：光镜下组织形态学观察结果及核素骨扫描结果示血管化程度是实验组优于对照组。 结果显示聚乳酸-聚羟基乙酸共聚物-成纤维细胞生长因子/磷酸钙骨水泥材料复合骨髓间充质干细胞构建的新型组织工程骨修复材料在动物体内有较好的成血管效果。     

Umbilical cord and bone marrow mesenchymal stem cell seeding on macroporous calcium phosphate for bone regeneration in rat cranial defects

Human umbilical cord mesenchymal stem cells (hUCMSCs) are inexhaustible and can be harvested at a low cost without an invasive procedure. However, there has been no report on comparing hUCMSCs with human bone marrow MSCs (hBMSCs) for bone regeneration in vivo. The aim of this study was to investigate hUCMSC and hBMSC seeding on macroporous calcium phosphate cement (CPC), and to compare their bone regeneration in critical-sized cranial defects in rats. Cell attachment, osteogenic differentiation and mineral synthesis on RGD-modified macroporous CPC were investigated in vitro. Scaffolds with cells were implanted in 8-mm defects of athymic rats. Bone regeneration was investigated via micro-CT and histological analysis at 4, 12, and 24 weeks. Three groups were tested: CPC with hUCMSCs, CPC with hBMSCs, and CPC control without cells. Percentage of live cells and cell density on CPC in vitro were similarly good for hUCMSCs and hBMSCs. Both cells had high osteogenic expressions of alkaline phosphatase, osteocalcin, collagen I, and Runx2. Bone mineral density and trabecular thickness in hUCMSC and hBMSC groups in vivo were greater than those of CPC control group. New bone amount for hUCMSC-CPC and hBMSC-CPC constructs was increased by 57% and 88%, respectively, while blood vessel density was increased by 15% and 20%, than CPC control group at 24 weeks. hUCMSC-CPC and hBMSC-CPC groups generally had statistically similar bone mineral density, new bone amount and vessel density. In conclusion, hUCMSCs seeded on CPC were shown to match the bone regeneration efficacy of hBMSCs in vivo for the first time. Both hUCMSC-CPC and hBMSC-CPC constructs generated much more new bone and blood vessels than CPC without cells. Macroporous RGD-grafted CPC with stem cell seeding is promising for craniofacial and orthopedic repairs.     

Orthodontic tooth movement in alveolar cleft repaired with a tissue engineering bone: an experimental study in dogs

Tissue engineering approaches have been successfully used in repairing bone defects and have become a viable alternative to autologous bone. The aim of the present study was to investigate if a construct of porous beta-tricalcium phosphate (β-TCP) combined with osteogenically induced bone marrow stromal cells (bMSCs) could repair alveolar cleft, and allow for subsequent orthodontic tooth movement in a canine model. Twelve alveolar osteotomy surgeries in six animals were made bilaterally and randomly implanted by (1) tissue-engineered bone complex of bMSCs/β-TCP (group A, n=4), (2) β-TCP alone (group B, n=4), and (3) autologous bone obtained from iliac bone (group C, n=4). Contralateral alveolar defects were created in one animal and left untreated to serve as blank control to observe spontaneous healing of the defects. Sequential fluorescent labeling and radiographic observation was used to evaluate new bone formation and mineralization in each defect. Orthodontic tooth movement was initiated 8 weeks after surgical operation for 12 weeks, and then the dogs were sacrificed for histological and histomorphometric analysis. Results indicated that the tissue-engineered complex with bMSCs/β-TCP dramatically promoted new bone formation and mineralization and achieved a favorable height of the repaired alveolar when compared with β-TCP alone, which absorbed severely. The overall effect of the tissue-engineered bone was equivalent to autologous bone; the physiological function of the alveolar bone was restored by allowing the adjacent teeth to move into the newly formed bone in the grafted region. This study demonstrated that the tissue engineering bone from the combination of β-TCP and bMSCs is a feasible clinical approach for patients with alveolar cleft and the subsequent orthodontic tooth movement.     

Tissue-engineered bone formation using periosteal-derived cells and polydioxanone/pluronic F127 scaffold with pre-seeded adipose tissue-derived CD146 positive endothelial-like cells

The aim of this study was to generate tissue-engineered bone formation using periosteal-derived cells seeded into a polydioxanone/pluronic F127 (PDO/Pluronic F127) scaffold with adipose tissue-derived CD146 positive endothelial-like cells. Considering the hematopoietic and mesenchymal phenotypes of adipose tissue-derived cells cultured in EBM-2 medium, CD146 positive adipose tissue-derived cells was sorted to purify more endothelial cells in characterization. These sorted cells were referred to as adipose tissue-derived CD146 positive endothelial-like cells. Periosteum is a good source of osteogenic cells for tissue-engineered bone formation. Periosteal-derived cells were found to have good osteogenic capacity in a PDO/Pluronic F127 scaffold, which could provide a suitable environment for the osteoblastic differentiation of these cells. Through the investigation of capillary-like tube formation on matrigel and the cellular proliferation of adipose tissue-derived CD146 positive endothelial-like cells cultured in different media conditions, we examined these cells could be cultured in EBM-2 with osteogenic induction factors. We also observed that the osteogenic activity of periosteal-derived cells could be good in EBM-2 with osteogenic induction factors, in the early period of culture. The experimental results obtained in the miniature pig model suggest that tissue-engineered bone formation using periosteal-derived cells and PDO/Pluronic F127 scaffold with pre-seeded adipose tissue-derived CD146 positive endothelial-like cells can be used to restore the bony defects of the maxillofacial region when used in clinics.     

Ectopic bone regeneration by human bone marrow mononucleated cells, undifferentiated and osteogenically differentiated bone marrow mesenchymal stem cells in beta-tricalcium phosphate scaffolds

Tissue engineering approaches using the combination of porous ceramics and bone marrow mesenchymal stem cells (BMSCs) represent a promising bone substitute for repairing large bone defects. Nevertheless, optimal conditions for constructing tissue-engineered bone have yet to be determined. It remains unclear if transplantation of predifferentiated BMSCs is superior to undifferentiated BMSCs or freshly isolated bone marrow mononucleated cells (BMNCs) in terms of new bone formation in vivo. The aim of this study was to investigate the effect of in vitro osteogenic differentiation (β-glycerophosphate, dexamethasone, and l-ascorbic acid) of human BMSCs on the capability to form tissue-engineered bone in unloaded conditions after subcutaneous implantation in nude mice. After isolation from human bone marrow aspirates, BMNCs were divided into three parts: one part was seeded onto porous beta-tricalcium phosphate ceramics immediately and transplanted in a heterotopic nude mice model; two parts were expanded in vitro to passage 2 before cell seeding and in vivo transplantation, either under osteogenic conditions or not. Animals were sacrificed for micro-CT and histological evaluation at 4, 8, 12, 16, and 20 weeks postimplantation. The results showed that BMSCs differentiated into osteo-progenitor cells after induction, as evidenced by the altered cell morphology and elevated alkaline phosphatase activity and calcium deposition, but their clonogenicity, proliferating rate, and seeding efficacy were not significantly affected by osteogenic differentiation, compared with undifferentiated cells. Extensive new bone formed in the pores of all the scaffolds seeded with predifferentiated BMSCs at 4 weeks after implantation, and maintained for 20 weeks. On the contrary, scaffolds containing undifferentiated BMSCs revealed limited bone formation only in 1 out of 6 cases at 8 weeks, and maintained for 4 weeks. For scaffolds with BMNCs, woven bone was observed sporadically only in one case at 8 weeks. Overall, this study suggests that ectopic osteogenesis of cell/scaffold composites is more dependent on the in vitro expansion condition, and osteo-differentiated BMSCs hold the highest potential concerning in vivo bone regeneration.     

Injectable tissue-engineered bone using autogenous bone marrow-derived stromal cells for maxillary sinus augmentation: clinical application report from a 2-6-year follow-up

This clinical study used injectable tissue-engineered bone, along with bone marrow-derived stromal cells (BMDSCs) and platelet-rich plasma (PRP), to conduct maxillary sinus floor augmentation by the simultaneous placement of bone graft and dental implants and to examine the state of regenerated bone after functional loading in 16 sinus augmentations in 12 patients whose alveolar crestal bone height was 2-10 mm. We used PRP as an autologous scaffold-which provides signal molecules-with in vitro expanded BMDSCs to enhance osteogenesis. All 41 dental implants prepared with the materials were clinically stable after second-stage surgery. The height of mineralized tissue at 2 years showed the mean increases of 8.8 +/- 1.6 mm compared to preoperative values, and no adverse effects and remarkable bone absorption were seen in the 2-6-year follow-up time. Although these results are preliminary, injectable tissue-engineered bone would stably predict the success of bone formation and dental implants, reduce patient burden, and provide minimally invasive cell therapy for patients.     

多孔磷酸钙骨水泥与转染骨形态发生蛋白2基因的骨髓间充质干细胞复合修复股骨髁骨缺损

BACKGROUND: Bone morphogenetic protein (BMP) can improve the osteogenesis capacity of tissue-engineered bone. However, how to prolong BMP release is a key for constructing tissue-engineered bone. OBJECTIVE: To study the repair effect of porous calcium phosphate cement (CPC) with bone marrow mesenchymal stem cells transfected with BMP-2 gene on bone defects. METHODS: After modeling of bilateral femoral condyle bone defects, 12 model rabbits were given implantation of porous CPC with bone marrow mesenchymal stem cells transfected with BMP-2 on the left (experimental group) and given implantation of porous CPC with bone marrow mesenchymal stem cells on the right (control group). Bilateral femoral condyles were taken and analyzed histologically at 4 and 12 weeks after implantation. RESULTS AND CONCLUSION: Better osteogenesis including more newly formed bone tissues and faster scaffold absorption was observed in the experimental group compared with the control group at 4 and 12 weeks after implantation. The area of newly formed bone tissues at different time and rate of bone formation at 12 weeks were significantly higher in the experimental group than in the control group (P < 0.001, P < 0.05). These findings indicate that transfer of BMP-2 into bone marrow mesenchymal stem cells combined with porous CPC could increase repair of bone defects.     

Human embryonic stem cell encapsulation in alginate microbeads in macroporous calcium phosphate cement for bone tissue engineering

Human embryonic stem cells (hESC) are promising for use in regenerative medicine applications because of their strong proliferative ability and multilineage differentiation capability. To date there have been no reports on hESC seeding with calcium phosphate cement (CPC). The objective of this study was to investigate hESC-derived mesenchymal stem cell (hESCd-MSC) encapsulation in hydrogel microbeads in macroporous CPC for bone tissue engineering. hESC were cultured to form embryoid bodies (EB), and the MSC were then migrated out of the EB. hESCd-MSC had surface markers characteristic of MSC, with positive alkaline phosphatase (ALP) staining when cultured in osteogenic medium. hESCd-MSC were encapsulated in alginate at a density of 1millioncellsml(-1), with an average microbead size of 207μm. CPC contained mannitol porogen to create a porosity of 64% and 218-μm macropores, with 20% absorbable fibers for additional porosity when the fibers degrade. hESCd-MSC encapsulated in microbeads in CPC had good viability from 1 to 21days. ALP gene expression at 21days was 25-fold that at 1day. Osteocalcin (OC) at 21days was two orders of magnitude of that at 1day. ALP activity in colorimetric p-nitrophenyl phosphate assay at 21days was fivefold that at 1day. Mineral synthesis by the encapsulated hESCd-MSC at 21days was sevenfold that at 1day. Potential benefits of the CPC-stem cell paste include injectability, intimate adaptation to complex-shaped bone defects, ease in contouring to achieve esthetics in maxillofacial repairs, and in situ setting ability. In conclusion, hESCd-MSC were encapsulated in alginate microbeads in macroporous CPC, showing good cell viability, osteogenic differentiation and mineral synthesis for the first time. The hESCd-MSC-encapsulating macroporous CPC construct is promising for bone regeneration in a wide range of orthopedic and maxillofacial applications.     

Repairing critical-sized calvarial defects with BMSCs modified by a constitutively active form of hypoxia-inducible factor-1α and a phosphate cement scaffold

Tissue engineering combined with gene therapy represents a promising approach for bone regeneration. The Hypoxia-inducible factor-1α (HIF-1α) gene is a pivotal regulator of vascular reactivity and angiogenesis. Our recent study has showed that HIF-1α could promote osteogenesis of bone mesenchymal stem cells (BMSCs) using a gene point mutant technique. To optimize the function of HIF-1α on inducing stem cells, another constitutively active form of HIF-1α (CA5) was constructed with truncation mutant method and its therapeutic potential on critical-sized bone defects was evaluated with calcium-magnesium phosphate cement (CMPC) scaffold in a rat model. BMSCs were treated with Lenti (lentivirus) -CA5, Lenti-WT (wild-type HIF-1α), and Lenti-LacZ. These genetically modified BMSCs were then combined with CMPC scaffolds to repair critical-sized calvarial defects in rats. The results showed that the overexpression of HIF-1α obviously enhanced the mRNA and protein expression of osteogenic markers in?vitro and robust new bone formation with the higher local bone mineral density (BMD) was found in?vivo in the CA5 and WT groups. Furthermore, CA5 showed significantly greater stability and osteogenic activity in BMSCs compared with WT. These data suggest that BMSCs transduced with truncation mutanted HIF-1α gene can promote the overexpression of osteogenic markers. CMPC could serve as a potential substrate for HIF-1α gene modified tissue engineered bone to repair critical sized bony defects.     

瓦楞状组织工程骨支架对种子细胞接种和成骨的影响

背景：不同形状工程骨支架负载种子细胞修复骨缺损的研究效果评价不一,而负载细胞数量的多少是影响疗效的重要因素之一,目前该方面研究证据不多。 目的：自制瓦楞状组织工程骨支架和其他3种形状的支架,比较4种不同形状支架负载种子细胞的数量,以及瓦楞状组织工程骨支架体内成骨时凹槽的优势及特点。 方法：①体外实验：将体积和样本数相同的4组支架分为单纯瓦楞状支架组、无瓦楞支架组、圆柱状支架组和带中空管柱状支架组,分别以相同密度、相同容积的成骨诱导兔骨髓间充质干细胞悬液接种于支架表面,孵育、培养、消化、收集,进行细胞计数、吸光度值检测以及碱性磷酸酶和茜素红染色。②体内实验：将兔随机分为重组人骨形态发生蛋白2/瓦楞状自固化磷酸钙人工骨组,瓦楞状自固化磷酸钙人工骨组和松质骨组,将体积相同的3组支架植入兔L5-6两侧横突间,植入后4,8,12周行大体、组织学观察。 结果与结论：体外实验显示,瓦楞状工程骨支架上滴注的细胞液能充分停留在表面,由于表面瓦楞状凹槽和液体的表面张力以及支架本身的无孔隙性使得细胞液不向培养皿流失,每个样本平均消化下来的正常形态的细胞数量高于其他3组(P < 0.05),吸光度值差异无显著性意义(P > 0.05)。体内实验显示,各时间点重组人骨形态发生蛋白2/瓦楞状自固化磷酸钙人工骨组的成骨量比瓦楞状自固化磷酸钙人工骨组明显多(P < 0.05),与松质骨组之间比较差异无显著性意义(P > 0.05)。结果证实,实验自制的瓦楞状工程骨支架的形状特点有利于种子细胞负载,从数量上保证了接种种子细胞的有效性,可促进支架大量成骨和段状骨缺损的愈合。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：