Stem cell technology and bioceramics: from cell to gene engineering

Mesenchymal stem cells reside in bone marrow and, when these cells are incorporated into porous ceramics, the composites exhibit osteo-chondrogenic phenotypic expression in ectopic (subcutaneous and intramuscular) or orthotopic sites. The expressional cascade is dependent upon the material properties of the delivery vehicle. Bioactive ceramics provide a suitable substrate for the attachment of the cells. This is followed by osteogenic differentiation directly on the surface of the ceramic, which results in bone bonding. Nonbioactive materials show neither surface-dependent cell differentiation nor bone bonding. The number of mesenchymal stem cells in fresh adult bone marrow is small, about one per one-hundred-thousand nucleated cells, and decreases with donor age. In vitro cell culture technology can be used to mitotically expand these cells without the loss of their developmental potency regardless of donor age. The implanted composite of porous ceramic and culture-expanded mesenchymal stem cells exhibits in vivo osteo-chondrogenic differentiation. In certain culture conditions, these stem cells differentiate into osteoblasts, which make bone matrix on the ceramic surface. Such in vitro prefabricated bone within the ceramic provides immediate new bone-forming capability after in vivo implantation. Prior to loading of the cultured, marrow-derived mesenchymal stem cells into the porous ceramics, exogenous genes can be introduced into these cells in culture. Combining in vitro manipulated mesenchymal stem cells with porous ceramics can be expected to effect sufficient new bone-forming capability, which can thereby provide tissue engineering approaches to patients with skeletal defects in order to regenerate skeletal tissues.     

Bone regeneration by grafting of cultured human bone

A 3-mL sample of bone marrow was collected from the iliac bones of 27 orthopedic patients (8 men and 19 women with a mean age of 56.1 years [range, 17 to 76 years]), followed by culture in standard culture medium (minimal essential medium containing 15% fetal bovine serum). In all 7 patients randomly selected from these 27 patients, significant in vitro osteogenic ability of marrow mesenchymal cells was demonstrated by scanning electron microscopy and biochemical analyses. In all 27 cases, to investigate the in vivo osteogenic potential of this human cultured bone, porous ceramics were impregnated with marrow cells and subcultured in osteogenic culture medium (standard medium supplemented with sodium beta-glycerophosphate, vitamin C phosphate, and dexamethasone). After 3 weeks of subculture, the cultured artificial bones of the cultured bone/porous ceramics were grafted into the abdominal cavity of nude mice. Histological and biochemical (alkaline phosphatase activity and human osteocalcin) examinations indicated that the cultured artificial bone possessed significant ability to regenerate bone. This result suggests that the bone-regenerating ability of human marrow cells may not depend on age, and that cultured artificial bone may be useful for bone regeneration treatment if appropriate cultured marrow cells can be successfully prepared.     

Growth of bone marrow cells on porous ceramics in vitro

Following the transplantation of bone marrow cells to extra-osseous sites, bone formation occurs in those sites. This osteogenic potential of bone marrow cells might be utilized for filling defects in bone if they could be transported on porous ceramic materials. Before such an approach becomes feasible, it is important to know what happens to the cells in the presence of the ceramics that might be used. In order to investigate the interaction between bone marrow cells and ceramics, in vitro, a system for culturing bone marrow cells on ceramic materials has been developed. Bone marrow cells adhered well to the surface of calcium hydroxyapatite and tricalcium phosphate ceramics, and this was followed by the formation of fibrous tissue on and within the ceramics. These ceramics were compatible with bone marrow cells even in culture conditions in which there was a large surface area of ceramic interfacing with cells. The results support the proposal that calcium hydroxyapatite and tricalcium phosphate are appropriate as bone replacement materials. In contrast, calcium aluminate had an adverse effect on bone marrow cells when there was a high proportion of ceramic to culture medium. However, this effect was not present if the proportion of ceramic to culture medium was low. Therefore, a large amount of biodegradable porous calcium aluminate ceramic should not be used as an alternative to autogeneous bone grafting.     

Osteogenic potential of culture-expanded rat marrow cells as assayed in vivo with porous calcium phosphate ceramic

It has been established that, when whole marrow is introduced into porous calcium phosphate ceramic, bone forms on the walls of the pores. To extend earlier studies, bone marrow cells derived from the femora of inbred rats were introduced into tissue culture and the adherent cells cultivated, mitotically expanded, passaged, harvested, placed in small cubes of porous calcium phosphate ceramics and grafted into subcutaneous sites of syngeneic rats. Marrow-derived, cultured mesenchymal cells introduced into ceramics showed strong osteogenic potential, with bone forming in the pore regions of ceramics as early as 2 wk after implantation. Osteogenesis could be observed after the eighteenth passage. With increasing passage number, the initiation of osteogenesis and the apparent rate of bone formation declined and the course of osteogenesis was delayed. In the future, it may be possible to culture marrow cells as a source for reparative cells for implantation back into autologous in vivo sites.     

Marrow cell induced osteogenesis in porous hydroxyapatite and tricalcium phosphate: a comparative histomorphometric study of ectopic bone formation

To investigate the bone formation ability of porous hydroxyapatite (HA) and tricalcium phosphate (TCP), ceramic discs were implanted with or without rat marrow cells into subcutaneous sites in syngeneic rats. The discs of HA and TCP had identical microstructures: pore size was 190-230 microns, porosity was 50-60%, and they were fully interconnected. Implants without marrow cells (discs themselves) did not show bone formation, whereas implants with marrow cells showed bone formation in the pores of the ceramics. The bone formation of both HA and TCP occurred initially on the surface of the ceramic and progressed towards the center of the pore. The de novo bone was quantitated from decalcified serial sections of the implants. One month after implantation with marrow cells, the percentage fractions of the pore area filled with bone for implanted HA and TCP were 16.9 and 15.1, respectively. At 2 months after implantation with marrow cells, the fractions of bone were 34.3 and 30.9, respectively. These results indicate that both HA and TCP ceramics can show comparable osteogenic ability in the presence of marrow cells.     

Flow Perfusion Culture of Marrow Stromal Cells Seeded on Porous Biphasic Calcium Phosphate Ceramics

Calcium phosphate ceramics have been widely used for filling bone defects to aid in the regeneration of new bone tissue. Addition of osteogenic cells to porous ceramic scaffolds may accelerate the bone repair process. This study demonstrates the feasibility of culturing marrow stromal cells (MSCs) on porous biphasic calcium phosphate ceramic scaffolds in a flow perfusion bioreactor. The flow of medium through the scaffold porosity benefits cell differentiation by enhancing nutrient transport to the scaffold interior and by providing mechanical stimulation to cells in the form of fluid shear. Primary rat MSCs were seeded onto porous ceramic (60% hydroxyapatite, 40% β-tricalcium phosphate) scaffolds, cultured for up to 16 days in static or flow perfusion conditions, and assessed for osteoblastic differentiation. Cells were distributed throughout the entire scaffold by 16 days of flow perfusion culture whereas they were located only along the scaffold perimeter in static culture. At all culture times, flow perfused constructs demonstrated greater osteoblastic differentiation than statically cultured constructs as evidenced by alkaline phosphatase activity, osteopontin secretion into the culture medium, and histological evaluation. These results demonstrate the feasibility and benefit of culturing cell/ceramic constructs in a flow perfusion bioreactor for bone tissue engineering applications.     

Substratum-dependent proliferation and survival of bone marrow-derived mononuclear phagocytes

Adherence to the culture substratum exerted a significant influence on the growth and survival of murine bone marrow cells incubated under conditions that favored the development of mononuclear phagocytes. Bone marrow cells cultured two weeks in unprocessed polystyrene dishes, refractory to cell attachment, reached a maximum or near maximum cell density by day 11. This density was stable throughout the duration of the incubation period. Characterization of the cells on day 11 revealed an essentially pure population of mononuclear phagocytes composed of round, nonadherent cells, and slightly elongated, loosely attached cells. Bone marrow cells incubated in polystyrene tissue culture dishes processed to promote cell attachment also reached a maximum cell density by day 11. However, this maximum density was only half that attained by cells incubated in unprocessed polystyrene dishes. Furthermore, continued incubation resulted in a sharp decline in cell viability and number. The cells in tissue culture dishes on day 11 represented a pure mononuclear phagocyte population composed principally of cells adherent to the surface of the dish. The subsequent analysis of cells subcultured in processed and unprocessed polystyrene dishes indicated that adherence to the substratum modulated the growth factor requirements of bone marrow-derived mononuclear phagocytes. Specifically, mononuclear phagocytes in tissue culture dishes expressed an elevated requirement for colony stimulating factor-1 in order to proliferate and survive in long-term culture.     

Analysis of primary bone formation in porous alumina: a fluorescence and scanning electron microscopic study of marrow cell induced osteogenesis.

Porous alumina ceramics alone and combined with rat marrow cells were implanted subcutaneously in the back of syngeneic Fischer rats. Fluorochrome-labeling was performed post operatively and the ceramics were harvested 6 and 8 weeks after implantation. Undecalcified sections of the implants were observed under fluorescence microscopy and the de novo bone-ceramic interfacial areas were analyzed by a scanning electron microscope equipped with an electron probe microanalyzer. Alumina ceramics alone did not show any bone formation, while all marrow cell loaded ceramics showed new bone formation 6 and 8 weeks after implantation. Bone formation was first observed in the center of the pores and proceeded in a centrifugal direction, leading to contact with the ceramic. These results suggest that bone marrow cells have inherent osteogenic capacity and in the pore region of alumina ceramics progression of the osteogenesis causes the dissipation of intervening fibrous tissue between the de novo bone and alumina ceramic surface.     

Osteogenic differentiation of cultured rat and human bone marrow cells on the surface of zinc-releasing calcium phosphate ceramics

Rat and human bone marrow cells (BMCs) were cultured on a composite ceramic of zinc-containing beta-tricalcium phosphate and hydroxyapatite (ZnTCP/HAP) with a (Ca+Zn)/P molar ratio of 1.60 and varying zinc contents. After a 2-week culture of the BMCs in the presence of beta-glycerophosphate and dexamethasone, many macroscopic mineralized areas with high alkaline phosphatase (ALP) activity were seen on the ZnTCP/HAP ceramic disks. The ALP activity increased with increasing zinc content in the ceramics. The highest ALP activity was observed when the BMCs were cultured on the ceramics with 1.26 wt % zinc, and the ceramics released zinc ions at concentrations from 2.2 to 7.2 microg/mL into the culture medium. Zinc ions were incorporated into mineralized areas produced by BMCs. BMCs also were directly cultured onto the culture dish surface, and the addition of 100 microM of free ZnCl(2) (6.5 microg/mL) to the culture medium significantly increased the ALP activity of the BMCs relative to the culture medium without the ZnCl(2)addition. The maximum zinc concentration required to enhance mineralization was higher in human BMCs than in rat BMCs. The present study demonstrates the superiority of ZnTCP/HAP ceramics over TCP/HAP in supporting the osteogenic differentiation of BMCs, and thus these ceramics are safe and useful in clinical settings, such as for bone reconstructive surgery.     

Hard tissue formation in a porous HA/TCP ceramic scaffold loaded with stromal cells derived from dental pulp and bone marrow

The aim of this study was to compare the ability of hard tissue regeneration of four types of stem cells or precursors under both in vitro and in vivo situations. Primary cultures of rat bone marrow, rat dental pulp, human bone marrow, and human dental pulp cells were seeded onto a porous ceramic scaffold material, and then either cultured in an osteogenic medium or subcutaneously implanted into nude mice. For cell culture, samples were collected at weeks 0, 1, 3, and 5. Results were analyzed by measuring cell proliferation rate and alkaline phosphatase activity, scanning electron microscopy, and real-time PCR. Samples from the implantation study were retrieved after 5 and 10 weeks and evaluated by histology and real-time PCR. The results indicated that in vitro abundant cell growth and mineralization of extracellular matrix was observed for all types of cells. However, in vivo matured bone formation was found only in the samples seeded with rat bone marrow stromal cells. Real-time PCR suggested that the expression of Runx2 and the expression osteocalcin were important for the differentiation of bone marrow stromal cells, while dentin sialophosphoprotein contributed to the odontogenic differentiation. In conclusion, the limited hard tissue regeneration ability of dental pulp stromal cells questions their practical application for complete tooth regeneration. Repeated cell passaging may explain the reduction of the osteogenic ability of both bone- and dentinal-derived stem cells. Therefore, it is essential to develop new cell culture methods to harvest the desired cell numbers while not obliterating the osteogenic potential.     

Three-dimensional engineered bone from bone marrow stromal cells and their autogenous extracellular matrix

Most bone tissue-engineering research uses porous three-dimensional (3D) scaffolds for cell seeding. In this work, scaffold-less 3D bone-like tissues were engineered from rat bone marrow stromal cells (BMSCs) and their autogenous extracellular matrix (ECM). The BMSCs were cultured on a 2D substrate in medium that induced osteogenic differentiation. After reaching confluence and producing a sufficient amount of their own ECM, the cells contracted their tissue monolayer around two constraint points, forming scaffold-less cylindrical engineered bone-like constructs (EBCs). The EBCs exhibited alizarin red staining for mineralization and alkaline phosphatase activity and contained type I collagen. The EBCs developed a periosteum characterized by fibroblasts and unmineralized collagen on the periphery of the construct. Tensile tests revealed that the EBCs in culture had a tangent modulus of 7.5 +/- 0.5 MPa at 7 days post-3D construct formation and 29 +/- 9 MPa at 6 weeks after construct formation. Implantation of the EBCs into rats 7 days after construct formation resulted in further bone development and vascularization. Tissue explants collected at 4 weeks contained all three cell types found in native bone: osteoblasts, osteocytes, and osteoclasts. The resulting engineered tissues are the first 3D bone tissues developed without the use of exogenous scaffolding.     

Observation of osteogenic differentiation cascade of living mesenchymal stem cells on transparent hydroxyapatite ceramics

The use of bioceramics and cultured cells for tissue engineering is a novel approach, which is available in a wide variety of clinical situations. The approach requires apparent verification of the cellular functions occurring on the ceramic surface, and these functions could be monitored by microscopic observation of the cultured living cells on the ceramic material. However, such observation is difficult due to the opaque nature of ordinary ceramics. To overcome this drawback, we used transparent hydroxyapatite (tHA) ceramics as a culture substrate and a transgenic rat having an enhanced green fluorescent protein (EGFP)-expressing gene as the cell source. Marrow mesenchymal stem cells (MSC) were obtained from the rat and cultured on both tHA ceramics and a tissue culture polystyrene (TCPS) dish. One hour after the cell seeding, many MSC had attached and showed initial cell spreading. The attachment and spreading were more obvious 5h after the seeding. Following the culture in the osteogenic condition, the cells differentiated into osteoblasts, which fabricated bone matrix on the culture substrate. The phenomena were similarly observed on both the tHA ceramics and TCPS substrata. These results confirm the excellent properties of tHA ceramics, which support cell attachment, proliferation, and differentiation. Transparent materials make us know the biological usefulness of ceramics in tissue-engineering field.     

Bonding osteogenesis in coralline hydroxyapatite combined with bone marrow cells

Coralline hydroxyapatite ceramics alone (control) and the ceramics combined with rat marrow cells were implanted subcutaneously in the backs of syngeneic Fischer rats and harvested at 1,2,3,4,6,8 and 24 wk after surgery. None of the control ceramics (without marrow) showed bone formation. However, ceramics combined with marrow cells showed consistent new bone formation in the pore regions. Histometrical results revealed increased new bone formation over time. Undecalcified sections of the ceramics studied by fluorochrome labelling showed that the osteogenesis began directly on the surface of the ceramic and proceeded centripetally towards the centre of the pores (bonding osteogenesis). SEM-EPMA analysis of the bone-ceramic interface also revealed direct bonding of bone to the ceramic surface.     

Cell based bone tissue engineering in jaw defects

In 6 patients the potency of bone tissue engineering to reconstruct jaw defects was tested. After a bone marrow aspirate was taken, stem cells were cultured, expanded and grown for 7 days on a bone substitute in an osteogenic culture medium to allow formation of a layer of extracellular bone matrix. At the end of the procedure, this viable bone substitute was not only re-implanted in the patient, but also simultaneously subcutaneously implanted in mice to prove its osteogenic potency. In all patients, a viable bone substitute was successfully constructed, which was proven by bone formation after subcutaneous implantation in mice (ectopic bone formation). However, the same construct was reluctant to form bone in patients with intra-oral osseous defects (orthotopic bone formation). Although biopsies, taken 4 months after reconstructing the intra-oral bone defect, showed bone formation in 3 patients, only in 1 patient bone formation was induced by the tissue-engineered construct. Although bone tissue engineering has proven its value in animal studies, extra effort is needed to make it a predictable method for reconstruction jaw defects in humans. To judge its benefit, it is important to differentiate between bone formation induced by cells from the border of the osseous defect (osteoconduction) in relation to bone matrix produced by the implanted cells (osteogenesis).     

煅烧骨的安全性

背景：经高温处理的煅烧骨具有类似自然骨的连续微孔结构,良好的生物相容性和降解性。 目的：观察牛煅烧骨的生物相容性、细胞相容性及毒性。 方法：①细胞相容性实验：将牛煅烧骨与第3 代已诱导的Wistar大鼠骨髓间充质干细胞复合培养。②溶血实验：将煅烧骨浸提液、生理盐水与双蒸水加入兔血中。③凝血实验：将煅烧骨加入兔血浆中。④急性毒性实验：在昆明种小鼠尾静脉分别注射煅烧骨浸提液、生理盐水。⑤微核实验：在小鼠腹腔分别注射煅烧骨浸提液、生理盐水与环磷酰胺。⑥局部刺激性实验：将煅烧骨浸提液、生理盐水分别注射于兔两侧脊柱皮下。⑦热源检测实验：在兔耳静脉注射煅烧骨浸提液。⑧皮下植入实验：将煅烧骨材料植入Wistar大鼠背部皮下。 结果与结论：煅烧骨材料无细胞毒性,具有良好的细胞及血液相容性;对皮肤、肌肉无刺激作用;对心、肝、肾重要器官无毒性作用;皮下植入后对周围组织无刺激作用,能够部分降解吸收并被机体组织替代;无致热作用,对凝血功能无影响,对小鼠骨髓细胞无抑制及毒性作用。     

In vitro osteogenic differentiation of rat bone marrow cells subcultured with and without dexamethasone

The aim of our study was to investigate the osteogenic potential of subcultured rat bone marrow cells. Rat bone marrow (RBM) cells were cultured with or without dexamethasone. Subsequently, osteogenic differentiation and expression was studied. When cells were cultured continuously in the presence of dexamethasone, cultures initially showed high alkaline phosphatase expression and abundant mineralization. Expression of differentiation markers decreased with passaging. After cells were passaged three times, no alkaline phosphatase activity and calcification were found. Primary cells cultured without dexamethasone showed low alkaline phosphatase and no calcification, and remained fibroblast-like. When these cells were subcultured in the presence of dexamethasone, the cells did show osteogenic differentiation. Nevertheless, this occurred at a significant lower level than with cells continuously cultured with dexamethasone. In addition, no differentiation was found after second passage. Our results indicate that subcultured undifferentiated RBM cells show osteogenic differentiation after addition of dexamethasone. Expression of alkaline phosphatase and mineralization is higher in cells continuously supplemented with dexamethasone. Still, even when dexamethasone is added continuously, RBM cells loose their osteogenic potential after several passages. Therefore, we conclude that subculture of undifferentiated rat bone marrow cells results in the loss of osteogenic potential of these cells.     

过表达P75神经生长因子受体负向调控骨髓间充质干细胞的成骨分化

文题释义： P75神经生长因子受体：即P75^NTR,神经生长因子的受体之一,以三聚体和单体的形式镶嵌于细胞表面,主要包含胞外区、跨膜区以及胞内区3个结构区域。低聚状态决定了其对细胞的主要调节功能,与配体结合后通过改变构象来进行信号传导,进而发挥调节细胞的作用。 成骨分化：多向分化干细胞向单能成骨细胞转变,并最终向骨组织发展的一系列生理过程称为成骨分化,对调节骨形成和骨再生具有重要意义,其成骨分化过程中,碱性磷酸酶的活力逐渐增加,细胞内或者胞外基质的钙等矿物质沉积,形成钙结节,骨钙素及Ⅰ型胶原蛋白表达增加。背景：P75^NTR广泛表达于神经组织及细胞中,并发挥着促进或抑制分化的双重作用;同时在骨折不愈合局部组织中也发现了P75^NTR存在着过表达,因此研究P75^NTR对骨髓间充质干细胞成骨分化的作用具有重要的意义,为临床上提高骨折不愈合修复的疗效提供重要靶点。 目的：观察P75^NTR过表达对大鼠骨髓间充质干细胞体外诱导成骨分化的影响。 方法：选取SD大鼠双侧股骨,用全骨髓分离贴壁法提取大鼠骨髓间充质干细胞并进行体外传代培养;构建表达EGFP的P75^NTR过表达质粒GV358-P75^NTR,并用空慢病毒载体包装收集P75^NTR过表达慢病毒载体;选取体外培养至原代10 d的大鼠骨髓间充质干细胞,消化后种板,加入P75^NTR过表达慢病毒及相关感染试剂进行感染实验,感染7 d后用荧光显微镜观察绿色荧光蛋白的表达,Western blot检测P75^NTR蛋白的过表达情况;感染后用常规培养基培养7 d,更换成骨诱导分化培养基培养,诱导培养后第7,10,14天用酶标法定量检测碱性磷酸酶活力,诱导培养后第7,14天用茜素红染色观察矿化结节形成情况。 结果与结论：①P75^NTR过表达慢病毒感染骨髓间充质干细胞后能表达出绿色荧光蛋白(感染效率约90%),且P75^NTR蛋白表达量明显增多,与阴性病毒对照组比较差异有显著性意义(P < 0.05),过表达P75^NTR细胞模型构建成功;②与阴性病毒对照组及未转染组相比,P75^NTR过表达组细胞诱导分化后相应时间点的碱性磷酸酶活力明显降低,矿化结节形成减少,细胞聚集分布减弱,差异有显著性意义(P < 0.05);③结果表明,P75^NTR过表达负向调控了大鼠骨髓间充质干细胞的成骨分化。局部组织中P75^NTR过表达抑制周围骨髓间充质干细胞成骨分化可能是骨缺损或骨折不愈合的重要因素。 ORCID: 0000-0001-6923-3177(朱伦井) 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     

Enhancement of tissue engineered bone formation by a low pressure system improving cell seeding and medium perfusion into a porous scaffold

To obtain more extensive bone formation in composites of porous ceramics and bone marrow stromal cells (BMSCs), we hypothesized that a low-pressure system would serve to facilitate the perfusion of larger number of BMSCs into the porous scaffold, enhancing bone formation within the composites. After culturing BMSCs in osteogenic medium, porous blocks of beta-tricalcium phosphate (beta-TCP) were soaked in the cell suspension. Composites of the block and BMSCs were put immediately into a vacuum desiccator. Low pressure was applied to the low pressure group, while controls were left at atmospheric pressure. Composites were incubated in vitro or subcutaneously implanted into syngeneic rats, then analyzed biologically and histologically. In the in vitro group, cell suspension volume, cell seeding efficiency, alkaline phosphatase (ALP) activity, and DNA content in the beta-TCP blocks were significantly higher in low pressure group than in the controls. Scanning electron microscopy (SEM) demonstrated that a greater number of cells covered the central parts of the composites in the low pressure group. ALP activity in the composites was increased at 3 and 6 weeks after implantation into rats. Histomorphometric analysis revealed more uniform and extensive bone formation in the low pressure group than in the controls. The application of low pressure during the seeding of BMSCs in perfusing medium into a porous scaffold is useful for tissue-engineered bone formation.     

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.     

Bone formation using novel interconnected porous calcium hydroxyapatite ceramic hybridized with cultured marrow stromal stem cells derived from Green rat

The clinical use of cultured marrow stromal stem cells (MSCs) has recently attracted attention in the field of tissue engineering. For the clinical use of the MSCs, a prominent scaffold is needed. A scaffold hybridized with MSCs is transformed into a "bioactive bone substitute," and this provides good osteoconduction. In this study, a novel calcium hydroxyapatite ceramic with an interconnected porous structure (IP-CHA) was used as a scaffold. MSCs were harvested from Green rats containing Green Fluorescent Protein (GFP), and then these hybrids were implanted into the tibias of Sprague-Dawley rats. The purposes of this study were to examine the osteogenic ability of these hybrids without coculture, and to evaluate whether the resulting bone formation originated from the grafted MSCs or the recipient's cells. The hybridized group showed excellent bone formation compared with the IP-CHA-only implant group. Observation of the implanted MSCs revealed that they survived 8 weeks after surgery, and differentiated into osteoblast-like cells, thus providing bone formation. This implantation of the MSCs/IP-CHA composite provides excellent osteoconduction, and is expected to have extensive clinical applications.