A collagen–phosphophoryn sponge as a scaffold for bone tissue engineering

Non-collagenous phosphoproteins that interact with a type-I collagen are thought to nucleate bone mineral into collagen networks of mineralized tissues. Previously, phosphophoryn cross-linked to type-I collagen was reported to be an effective nucleator of appatite. However, free phosphophoryn molecules inhibit the formation of apatite in vitro. On the basis of the above study, we expected a collagen-phosphophoryn sponge to be a good scaffold for bone-tissue engineering and examined the formation of bone in orthotopically transplanted composites of the sponge and bone marrow osteoblasts in vivo in Fischer rats. Osteoblastic primary cells were obtained from the bone shaft of femorae of Fisher rats, according to the method of Maniatopoulous et al. A suspension of marrow cells was distributed through a flask with standard culture medium and incubated at 37°C. When cultures were nearly confluent after 10 days, they were concentrated by centrifugation to 10⁶ cells/ml and subcultured onto the synthesized collagen-phosphophoryn sponge and a collagen sponge (control). After 14 days, the composites of collagen-phosphophoryn and osteoblastic cells as well as control composites were transplanted into bone-defect sites of Fisher rats (holes 2 mm in diameter) and then the wounds were sutured. The composites were harvested at 1-8 weeks after implantation, and stained with hematoxylin and eosin. It was found that more bone was formed in the composites of collagen-phosphophoryn sponge and osteoblasts than control composites from 1 week to 8 weeks, suggesting that the collagen-phosphophoryn sponge is a good candidate as a scaffold for bone-tissue engineering.     

胶原-纳米羟基磷灰石复合支架的细胞相容性

背景：观察成骨细胞在生物材料上的形态、增殖和分化等项目,可评估生物支架材料的生物相容性。 目的：观察复合支架材料纳米羟基磷灰石/胶原对成骨细胞增殖、分化的影响。 方法：取新生24 h内Wistar大鼠的颅盖骨,采用改良胶原酶消化法进行成骨细胞原代培养,取第3代细胞与纳米羟基磷灰石/胶原支架或普通羟基磷灰石材料体外复合培养。培养3,6,9 d后,观察材料周边的细胞形态及支架材料对细胞分化、增殖的影响。 结果与结论：纳米羟基磷灰石/胶原材料较普通的羟基磷灰石材料更有利于成骨细胞的黏附、生长、分化、增殖,证实其生物相容性更好,有望成为一种新型的骨组织工程支架材料。     

Efficient Bone Regeneration Induced by Bone Morphogenetic Protein-2 Released from Apatite-Coated Collagen Scaffolds

Abstract

Bone morphogenetic proteins (BMPs) are the most potent osteoinductive growth factors. Clinically utilized BMP-2 uses a type-I collagen scaffold as a carrier. Here we hypothesized that an apatite coating on a type-I collagen scaffold would prolong the BMP-2 release period and enhance bone regeneration in calvarial defects in mice. Apatite coating was achieved by incubating collagen scaffolds in simulated body fluid. BMP-2 release kinetics and bioactivity were evaluated by enzyme-linked immunosorbent assay and alkaline phosphatase activity measurement of cultured osteoblasts. Computed tomography and histomorphometry were performed eight weeks after various doses of BMP-2 were delivered to mouse calvarial defects using either non-modified or apatite-coated collagen scaffolds. Apatite-coated collagen scaffolds released 91.8 ± 11.5% of the loaded BMP-2 over 13 days in vitro, whereas non-modified collagen scaffolds released 98.3 ± 2.2% over the initial one day. The in vivo study showed that BMP-2 delivery with apatite-coated collagen scaffolds resulted in a significantly greater bone formation area and higher bone density than that with non-modified collagen scaffolds. This study suggests that simple apatite coating on collagen scaffolds can enhance the bone regeneration efficacy of BMP-2 released from collagen scaffolds.     

Performance of collagen sponge as a 3-D scaffold for tooth-tissue engineering

Tooth structure can be regenerated by seeding dissociated tooth cells onto polyglycolic acid fiber mesh, although the success rate of tooth production is low. The present study was designed to compare the performance of collagen sponge with polyglycolic acid fiber mesh as a 3-D scaffold for tooth-tissue engineering. Porcine third molar teeth at the early stage of crown formation were enzymatically dissociated into single cells, and the heterogeneous cells were seeded onto collagen sponge or the polyglycolic acid fiber mesh scaffolds. Scaffolds were then cultured to evaluate cell adhesion and ALP activity in vitro. An in vivo analysis was performed by implanting the constructs into the omentum of immunocompromised rats and evaluating tooth production up to 25 weeks. After 24h, there were a significantly higher number of cells attached to the collagen sponge scaffold than the polyglycolic acid fiber mesh scaffold. Similarly, the ALP activity was significantly higher for the collagen sponge scaffold was than the polyglycolic acid fiber mesh scaffold after 7 days of culture. The area of calcified tissue formed in the collagen sponge scaffold was also larger than in the polyglycolic acid fiber mesh scaffold. The results from in vivo experiments show conclusively that a collagen sponge scaffold allows tooth production with a higher degree of success than polyglycolic acid fiber mesh. Taken together, the results from this study show that collagen sponge scaffold is superior to the polyglycolic acid fiber mesh scaffold for tooth-tissue engineering.     

Acceleration of bone formation with BMP2 in frame-reinforced carbonate apatite–collagen sponge scaffolds

The development is expected of scaffold biomaterials that feature a shape-maintaining property in addition to high porosity and large pores that cells can easily invade. To develop a new biodegradable scaffold biomaterial reinforced with a frame, synthesized carbonate apatite (CO₃Ap) was mixed with neutralized collagen gel, and the CO₃Ap–collagen mixtures were lyophilized into sponges in a porous hydroxyapatite (HAp) frame ring. X-ray diffraction and Fourier transform infrared spectroscopy (FT-IR) analyses together with chemical analysis indicated that the synthesized CO₃Ap had a crystalline nature and a chemical composition similar to that of bone. Scanning electron microscope (SEM) observation showed that the CO₃Ap–collagen sponge had a sui pore size for cell invasion. In proliferation and differentiation experiments with osteoblasts, alkaline phosphatase and osteopontin activity were clearly detected. When these sponge–frame complexes with bone morphogenic protein (rh-BMP2) were implanted beneath the periosteum cranii of rats, significant new bone was created at the surface of the periosteum cranii after 4 weeks of implantation. These reinforced CO₃Ap–collagen sponges with rh-BMP2 are expected to be used as hard tissue scaffold biomaterials for the therapeutic purpose of the rapid cure of bone defects.     

胶原海绵上诱导骨髓间充质干细胞向软骨细胞分化的体外实验研究

研究在胶原海绵上间充质干细胞(M SC s)向软骨细胞的定向诱导,以及其对M SC s细胞增殖力、细胞周期的影响,为临床应用胶原海绵作为M SC s支架、修复软骨缺损奠定基础。取大鼠股骨骨髓,经梯度离心法和贴壁法分离M SC s,用加有转化生长因子1β10 ng/m l、地塞米松1-0 7m o l/L、转铁蛋白3 m g/m l、胰岛素2 m g/m l的诱导因子培养基分别在胶原海绵、培养板中进行诱导,14 d后免疫组化检测II型胶原分泌情况,四甲基偶氮唑蓝(M TT)法比较两者之间细胞增殖情况,流式细胞仪(FCM)检测各组细胞周期。14 d后,胶原海绵上及培养板中均诱导出表达II型胶原的软骨细胞,M TT法检测胶原海绵诱导组,和培养板诱导组细胞增殖值分别为0.9213±0.0312和0.5875±0.0258,两者间有显著性差异(P<0.05),FCM检测胶原海绵各组S期 G2/M期百分率,发现其与培养板中同期细胞相比有显著性差异(P<0.05),各组均未发现异倍体。结果表明在胶原海绵上,M SC s能定向诱导为软骨细胞,细胞增值能力更强,M SC s细胞DNA合成、细胞分裂增加,无异倍体产生,可作为一种较为理想的组织工程学支架。     

Bone regeneration on a collagen sponge self-assembled peptide-amphiphile nanofiber hybrid scaffold

The objective of this study was to create a novel approach to promote bone induction through sustained release of growth factor from a 3-dimensional (3D) hybrid scaffold. Peptide-amphiphile (PA) was synthesized by standard solid-phase chemistry that ends with the alkylation of the NH2 terminus of the peptide. Collagen sponge was reinforced by incorporation of poly(glycolic acid) (PGA) fiber. A 3D network of nanofibers was formed by mixing basic fibroblast growth factor (bFGF) suspensions with dilute aqueous solutions of PA. A hybrid scaffold was fabricated by combination of self-assembled PA nanofibers and collagen sponge reinforced with incorporation of PGA fibers. The in vitro release profile of bFGF from hybrid scaffold was investigated, and ectopic bone formation induced by the released bFGF was assessed after subcutaneous implantation of hybrid scaffold into the backs of rats. Homogeneous bone formation was histologically observed throughout the hybrid scaffolds, in marked contrast to collagen sponge-incorporated bFGF. The level of alkaline phosphatase activity and osteocalcin content at the implanted sites of hybrid scaffolds were significantly high compared with collagen sponge incorporated with bFGF. The combination of bFGF incorporated in a collagen sponge self-assembled PA nanofiber hybrid scaffold is a promising procedure to improve bone regeneration.     

腺病毒携带骨形态发生蛋白14基因转染脂肪干细胞修复损伤关节软骨

背景：关节软骨损伤后自我修复能力较弱,主要是由于其缺乏滋养血管并且细胞代谢缓慢等组织特性,目前的治疗方法都不能恢复软骨组织的原有功能,近年来软骨组织工程已引起了越来越多的关注。 目的：观察Ⅰ型胶原海绵支架搭载骨形态发生蛋白14基因转染脂肪干细胞修复兔膝关节软骨损伤的效果。 方法：取兔皮下脂肪组织分离培养脂肪干细胞,用腺病毒真核表达载体Ad-CMV-BMP-14-IRES-hrGFP-1转染脂肪干细胞。Ⅰ型胶原海绵支架搭载转染后的脂肪干细胞,待细胞吸附后对兔膝关节全层软骨缺损进行修复。术后12周取手术关节,从大体方面、组织学方面综合评估缺损修复状况。 结果与结论：骨形态发生蛋白14转染后的脂肪干细胞骨形态发生蛋白14和Ⅱ型胶原蛋白表达及Sox-9基因表达明显高于普通脂肪干细胞。术后12周,支架搭载经骨形态发生蛋白14转染的脂肪干细胞组软骨组织修复良好,平整光滑,光洁度、质地及颜色良好,交界区整合良好。支架搭载脂肪干细胞组软骨组织部分修复,有正常软骨光泽,质地与颜色接近正常,修复组织与正常软骨组织界限明显。单纯支架组几乎崩解塌陷,未见透明样软骨结构形成。结果可见腺病毒携带骨形态发生蛋白14基因转染后脂肪干细胞修复软骨缺损的能力有大幅提升。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

多孔碳酸钙陶瓷与干细胞源性成骨细胞的相容性

背景：国内外的研究证实普通碳酸钙陶瓷作为骨替代材料时具有细胞支架作用。 目的：观察多孔碳酸钙陶瓷与成骨细胞的相容性,及作为骨组织工程支架的可能性。 方法：SD大鼠骨髓基质干细胞经矿化诱导培养、扩增并检测证实其已具成骨细胞表型后,分别与多孔碳酸钙陶瓷支架、普通羟基磷灰石陶瓷支架体外复合培养。 结果与结论：骨髓基质干细胞经体外诱导形成成骨细胞,钙结节、Ⅰ型胶原和碱性磷酸酶免疫染色结果阳性。多孔碳酸钙陶瓷支架材料与羟基磷灰石陶瓷材料皆有细胞附着生长,但多孔碳酸钙陶瓷支架材料细胞的黏附能力、增殖活力及成骨活性均强于羟基磷灰石陶瓷材料。提示多孔碳酸钙陶瓷支架材料与SD大鼠骨髓基质干细胞源性成骨细胞有良好相容性。     

胶原海绵支架体外构建组织工程膀胱黏膜***◆

背景：组织工程膀胱黏膜层的构建在组织工程膀胱修复中占有重要的地位,但目前并没有最合理的构建方法。 目的：探讨胶原海绵支架复合猪膀胱尿路上皮细胞体外构建膀胱黏膜结构的可行性。 方法：刮取猪膀胱黏膜层后用酶消化方式进行猪膀胱尿路上皮细胞原代培养,并进行尿路上皮细胞标志物免疫荧光和RT-PCR鉴定。制备疏松多孔的胶原海绵支架材料,将第3代尿路上皮细胞接种在胶原海绵支架上,体外培养4~8 d后观察尿路上皮细胞和胶原海绵材料的复合情况。 结果与结论：原代培养的猪膀胱尿路上皮细胞呈“多角形”、“铺路石”样,以克隆团形式生长。免疫荧光鉴定AE1/AE3阳性,RT-RCR检测uroplakin-ⅠA、uroplakin-Ⅱ阳性。胶原海绵复合尿路上皮细胞体外培养4~8 d后,细胞在胶原海绵支架上生长良好,覆盖胶原材料的表面并长入材料内部,保持了尿路上皮细胞的特性。体外培养6 d时,尿路上皮细胞与胶原支架复合效果最好,同时细胞数量也最多。结果初步表明了胶原海绵复合膀胱尿路上皮细胞可以构建组织工程膀胱黏膜,且体外培养6 d为最佳时间点。     

Use of a biomimetic strategy to engineer bone

Engineering trabecular-like, three-dimensional bone tissue throughout biodegradable polymer scaffolds is a significant challenge. Using a novel processing technique, we have created a biodegradable scaffold with geometry similar to that of trabecular bone. When seeded with bone-marrow cells, new bone tissue, the geometry of which reflected that of the scaffold, was evident throughout the scaffold volume and to a depth of 10 mm. Preseeded scaffolds implanted in non-healing rabbit segmental bone defects allowed new functional bone formation and bony union to be achieved throughout the defects within 8 weeks. This marks the first report of successful three-dimensional bone-tissue engineering repair using autologous marrow cells without the use of supplementary growth factors. We attribute our success to the novel scaffold morphology.     

Histological and biomechanical properties of regenerated articular cartilage using chondrogenic bone marrow stromal cells with a PLGA scaffold in vivo

The properties of regenerated cartilage using bone marrow-derived mesenchymal stem cells (MSCs) and poly lactic-co-glycolic acid (PLGA) scaffold composites pretreated with TGF-beta3 were investigated and compared to the non-TGF-beta3 treated MSCs/PLGA composites in a rabbit model. We prepared MSCs/PLGA scaffold composites and pretreated it with TGF-beta3 for 3 weeks prior to transplantation. Then, composites were transplanted to the osteochondral defect in the rabbit knee. After 12 weeks of transplantation, 10 of the 12 rabbits in which TGF-beta3 pretreated MSCs/PLGA scaffold composites were transplanted showed cartilaginous regeneration. In gross morphology, regenerated cartilage showed smooth, flush, and transparent features. In indentation test, this had about 80% of Young's modulus of normal articular cartilage. Histological examination demonstrated hyaline like cartilage structures with glycosaminoglycan and type II collagen expression. Histological scores were not statistically different to the normal articular cartilage. These results showed improvement of cartilage regeneration compared to the non-TGF-beta3 pretreated MSCs/PLGA scaffold composite transplanted group. Thus, we have successfully regenerated improved hyaline-like cartilage and determined the feasibility of treating damaged articular cartilage using MSCs/PLGA scaffold composite pretreated with TGF-beta3. Also, we suggest this treatment modality as another concept of cartilage tissue engineering.     

Reinforcement of a Porous Collagen Scaffold with Surface-Activated PLA Fibers

A hybrid porous collagen scaffold mechanically reinforced with surface-activated poly(lactic acid) (PLA) fiber was prepared. PLA fibers, 20 μm in diameter and 1 mm in length, were aminolyzed with hexanediamine to introduce free amino groups on the surfaces. After the amino groups were transferred to aldehyde groups by treatment with glutaraldehyde, different amounts (1.5, 3, 5 and 8 mg) of surface-activated PLA fibers were homogeneously mixed with 2 ml type-I collagen solution (pH 2.8, 0.6 wt%). This mixture solution was then freeze-dried and cross-linked to obtain collagen sponges with surface-activated PLA fiber. Scanning electron microscopy observation indicated that the collagen sponges had a highly interconnected porous structure with an average pore size of 170 μm, irrespective of PLA fiber incorporation. The dispersion of surface-activated PLA fibers was homogeneous in collagen sponge, in contrast to unactivated PLA fibers. The compression modulus test results showed that, compared with unactivated PLA fibers, the surface-activated PLA fibers enhanced the resistance of collagen sponge to compression more significantly. Cytotoxicity assay by MTT test showed no cytotoxicity of these collagen sponges. L929 mouse fibroblast cell-culture studies in vitro revealed that the number of L929 cells attached to the collagen sponge with surface-activated PLA fibers, both 6 h and 24 h after seeding, was higher than that in pure collagen sponge and sponge with unactivated PLA fibers. In addition, a better distribution of cells infiltrated in collagen sponge with surface-activated PLA fibers was observed by histological staining. These results indicated that the collagen sponge reinforced with surface-activated PLA fibers is a promising biocompatible scaffold for tissue engineering.     

大鼠骨髓间充质干细胞的体外成骨诱导*

背景：骨髓间充质干细胞作为良好的种子细胞,将其复合于生物支架治疗骨缺损取得了良好的进展,是当今研究的一大热点。 目的：观察大鼠骨髓间充质干细胞体外诱导向成骨样细胞分化的效果。 方法：采用贴壁筛选法分离培养SD大鼠骨髓间充质干细胞,取生长状态良好的第3代骨髓间充质干细胞,分为2组,对照组细胞仅用DMEM/F12培养基培养;实验组以含成骨诱导剂的DMEM/F12培养基培养。 结果与结论：全骨髓贴壁法培养的原代骨髓间充质干细胞呈梭形或多角形贴壁生长;经成骨诱导剂诱导后骨髓间充质干细胞呈圆形或卵圆形贴壁生长,碱性磷酸酶活性明显强于对照组(P < 0.01);茜素红染色出现阳性的钙化结节;Western blotting检测Ⅰ型胶原蛋白表达较对照组有明显增加(P < 0.01);骨钙素ELISA定量分析较对照组明显升高(P < 0.01)。提示全骨髓贴壁法培养骨髓间充质干细胞方法简单、实用,所培养的骨髓间充质干细胞在成骨诱导后表现了成骨细胞的形态学和生物学特性。     

兔骨髓成骨细胞同种异体移植免疫反应的初步观察

以观察新生兔骨髓诱导分化为成骨细胞的能力 ,探讨同种异体成骨细胞移植的可行性 ,为进一步的研究奠定基础。取新生新西兰大白兔胫骨骨髓 ,分离后加入条件培养液体外培养。传 5代后对细胞进行形态学、碱性磷酸酶 (ALP )染色及体外矿化能力的检测。将冻存复苏的细胞以明胶海绵吸附 ,植入异体成年兔皮下、肌肉内 ,第 2、 4、 8、 12周时取材观察 ,分析其成骨能力及免疫排斥反应情况。结果显示体外诱导培养的骨髓成骨细胞是一较纯的细胞系 ,以带突起的梭形细胞为主 ,ALP染色阳性 ,连续培养 4 0d可见矿化结节形成。异体植入的成骨细胞大部分存活 ,4周后开始有类骨基质形成 ,并可见不规则的矿化骨组织 ,植入细胞周围仅见少量的淋巴细胞和嗜酸粒细胞浸润。骨髓基质细胞具有多向分化的潜力 ,异体植入的细胞仍保持基本的生物学功能 ,免疫排斥反应比较轻微 ,提示细胞异体移植是可行的     

Influence of the in vitro culture period on the in vivo performance of cell/titanium bone tissue-engineered constructs using a rat cranial critical size defect model

The aim of this study was to investigate the in vivo performance in bone-regenerating capability of cell/scaffold constructs implanted into an orthotopic site. Bone marrow stromal osteoblasts were seeded on titanium fiber mesh scaffolds using a cell suspension (5 x 10(5) cells per scaffold) and cultured for 1, 4, and 8 days under either static or flow perfusion conditions forming six different treatment groups. A total of 16 constructs from each one of the six treatment groups were then implanted into an 8-mm critical size calvarial defect created in the cranium of adult syngeneic male Fisher rats. Half of the constructs from each group were retrieved 7 days postimplantation, and the other half of the constructs were retrieved 30 days postimplantation and examined for new bone formation and tissue response. Constructs retrieved 7 days postimplantation were filled with fibrous tissue and capillaries, but no bone formation was observed in any of the six treatment groups. Constructs retrieved 30 days postimplantation showed bone formation (at least 7 out of 8 constructs in all treatment groups). Titanium fiber meshes seeded with bone marrow stromal osteoblasts and cultured for 1 day under flow perfusion conditions before implantation appeared to give the highest percentage of bone formation per implant (64 +/- 17%). They also showed the highest ratio of critical size cranial defects that resulted in union of the defect 30 days postimplantation (7 out of 8) together with the constructs cultured for 1 day under static conditions before implantation. There were no significant differences between the different treatment groups; this finding is most likely due to the large variability of the results and the small number of animals per group. However, these results show that titanium fiber mesh scaffolds loaded with bone marrow stromal osteoblasts can have osteoinductive properties when implanted in an orthotopic site. They also indicate the importance of the stage of the osteoblastic differentiation and the quality of the in vitro generated extracellular matrix in the observed osteoinductive potential.     

A platelet-derived growth factor releasing chitosan/coral composite scaffold for periodontal tissue engineering

In recent years, functional biomaterial research has been directed towards the development of improved scaffolds and new drug delivery systems. The objective of this study was to develop growth-factor gene releasing coral composites as a regenerative material for periodontal regeneration. In this study, porous chitosan/coral composites combined with plasmid encoding platelet-derived growth factor B (PDGFB) gene were prepared through a freeze-drying process. These scaffolds were evaluated in vitro by analysis of microscopic structure and cytocompatibility. The expression of PDGFB and type-I collagen were detected with RT-PCR after human periodontal ligament cells (HPLCs) were seeded in this scaffold. Then these scaffolds were implanted subcutaneously into athymic mice. Results indicated that HPLCs showed much better proliferation properties on the gene-activated scaffolds than on the pure coral scaffolds, and the expression of PDGFB and type-I collagen up-regulated in gene-activated scaffold. After implanted in vivo, HPLCs not only proliferate but also increased the expression of PDGFB. This study demonstrated the potential of coral scaffold combined PDGFB gene as a good substrate candidate in periodontal tissue regeneration.     

In vitro ossification and remodeling of mineralized collagen I scaffolds

A promising strategy of bone tissue engineering is to repair bone defects by implanting biodegradable scaffolds that can undergo remodeling and be replaced completely by autologous bone tissue. For this purpose, it is necessary to create scaffolds that can be degraded by osteoclasts and enable osteoblasts to build new mineralized bone matrix. In order to achieve this goal a new porous material has been developed using biomimetically mineralized collagen I. These scaffolds were co-cultured with osteoclast-like cells and osteoblasts in order to characterize the capacity of these cells to remodel the material in vitro. It was possible to show the development of biologically active osteoclast- like cells that were able to invade and degrade the scaffold. They degraded the scaffold by internalizing it as intracellular vesicles, thereby making room for osteoblasts to invade and build new bone matrix. In addition, it could be shown that osteoblasts proliferated, differentiated, and produced new mineralized extracellular matrix. Hence, it could be shown that co-culture of osteoclastlike cells and osteoblasts on biomimetically mineralized collagen I is a promising approach for bone tissue engineering. In addition, it can be applied to study the process of bone remodeling in vitro.     

以带血管蒂筋膜瓣与人脐带间充质干细胞和重组人骨形成蛋白2构建活性组织工程骨

背景：构建的组织工程骨块植入体内后的存活是骨组织工程面临的一个重大课题,临床上尤其缺少可行性强、可以不经体外长时间构建及预血管化而可一期应用的组织工程骨。 目的：探讨以带血管蒂的筋膜瓣作为膜包裹材料、以人脐带间充质干细胞作为种子细胞,以β-磷酸三钙生物陶瓷作为支架材构建组织工程骨的可行性及加入重组人骨形成蛋白2作为细胞活性因子、Ⅰ型胶原作为细胞活性因子缓释材料后成骨能力的变化。 方法：Wistar大鼠左侧L1~6背部带血管蒂的筋膜瓣包绕由β-磷酸三钙生物陶瓷、人脐带间充质干细胞、重组人骨形成蛋白2、Ⅰ型胶原构建的组织工程骨作为实验侧,右侧带血管蒂的筋膜瓣包绕接种了人脐带间充质干细胞的β-磷酸三钙生物陶瓷作为对照侧。 结果与结论：大鼠实验侧4周时幼稚骨组织连接形成原始层板骨样结构,形成的原始骨组织钙化程度低。8周时,大鼠两侧骨组织均基本成熟。成骨细胞位于骨陷窝中,周围有大量骨基质呈淡紫色,局部可见Ⅰ型胶原存在,有骨髓腔结构出现,但实验侧骨组织成熟度明显高于对照侧。实验侧骨组织哈夫氏小管清晰可见,形成多个骨化中心,骨小梁、骨岛遍布其中,可见成熟板层骨、立方状排列整齐的活性成骨细胞。8周时实验侧骨小梁成熟度高、典型、清晰可见,对照侧骨小梁成熟度稍差。但2组植入物的新骨形成面积接近。提示以带血管蒂的筋膜瓣作为膜包裹材料构建组织工程骨时,重组人骨形成蛋白2及缓释剂Ⅰ型胶原可促进其骨成熟度。     

Enhancement of the in vivo osteogenic potential of marrow/hydroxyapatite composites by bovine bone morphogenetic protein

A composite of marrow mesenchymal stem cells and porous hydroxyapatite (HA) has in vivo osteogenic potential. To investigate factors enhancing the osteogenic potential of marrow/HA composites, we prepared a bone morphogenetic protein (BMP) fraction from the 4M guanidine extract of bovine bone by heparin-sepharose affinity chromatography. Marrow/HA composites or composites containing marrow mesenchymal stem cells, BMP, and HA (marrow/BMP/HA composites) were implanted subcutaneously in 7-week-old male Fischer rats. BMP/HA composites and HA alone were also implanted. The implants were harvested after 2, 4, or 8 weeks and were prepared for histological and biochemical studies. Histological examination showed obvious de novo bone formation together with active osteoblasts at 2 weeks, as well as more extensive bone formation at 4 and 8 weeks in many pores of the marrow/BMP/HA composites. The marrow/HA composites did not induce bone formation at 2 weeks, but there was moderate bone formation at 4 weeks. At 2 weeks, only marrow/BMP/HA composites resulted in intensive osteogenic activity, judging from alkaline phosphatase and osteocalcin expression at both the protein and gene levels. These results indicate that the combination of marrow mesenchymal stem cells, porous HA, and BMP synergistically enhances osteogenic potential, and may provide a rational basis for their clinical application, although further in vivo experiment is needed.