Bone formation by isolated calvarial osteoblasts in syngeneic and allogeneic transplants: Light microscopic observations

Taking advantage of recently developed methods for osteoblast isolation, we used these cells to study bone morphogenesis in syngeneic and allogeneic intramuscular transplants. Syngeneic osteoblasts from fetal rat calvaria produced small islands of bone by the third day after transplantation. These islands increased in size and began to fuse after about 14 days. At the surface of the woven bone laid down first, lamellar bone developed. The amount of this bone increased, and in 56-day-old transplants solid blocks of bone were present. Osteoclasts were scarce, and the woven bone remained unresorbed. Bone marrow was absent. The structure of bone in transplants differed from that of mature calvarial bones in which only remnants of woven bone remained and bone marrow was well developed. The scarcity of osteoclasts in transplants could be caused by their relative paucity among the injected cells, since these cells responded strongly to added parathyroid hormone by increased production of cyclic adenosine monophosphate (cAMP) but only weakly to calcitonin. Osteoblasts isolated from the surface of calvarial lamellar bone of 28-day-old rats formed woven bone similar to the bone formed by fetal cells. This suggests that the type of bone produced does not depend on the intrinsic properties of the osteoblasts. Bone formed in an allogeneic system was surrounded by infiltrations containing lymphocytes, macrophages, and osteoclastlike cells in 14-day-old transplants. Osteoblasts at the bone periphery were destroyed and bone matrix was resorbed by infiltrating cells. Numerous bone lacunae were enlarged, suggesting the occurrence of osteocytic osteolysis. Isolated osteoblasts cultured for three population doublings or longer did not form bone after transplantation, although they retained some reactivity toward parathyroid hormone.     

Engineering endochondral bone: in vivo studies

The use of biomaterials to replace lost bone has been a common practice for decades. More recently, the demands for bone repair and regeneration have pushed research into the use of cultured cells and growth factors in association with these materials. Here we report a novel approach to engineer new bone using a transient cartilage scaffold to induce endochondral ossification. Chondrocyte/chitosan scaffolds (both a transient cartilage scaffold-experimental-and a permanent cartilage scaffold-control) were prepared and implanted subcutaneously in nude mice. Bone formation was evaluated over a period of 5 months. Mineralization was assessed by Faxitron, micro computed tomography, backscatter electrons, and Fourier transform infrared spectroscopy analyses. Histological analysis provided further information on tissue changes in and around the implanted scaffolds. The deposition of ectopic bone was detected in the surface of the experimental implants as early as 1 month after implantation. After 3 months, bone trabeculae and bone marrow cavities were formed inside the scaffolds. The bone deposited was similar to the bone of the mice vertebra. Interestingly, no bone formation was observed in control implants. In conclusion, an engineered transient cartilage template carries all the signals necessary to induce endochondral bone formation in vivo.     

Expression of bone morphogenetic proteins and cartilage-derived morphogenetic proteins during osteophyte formation in humans

Bone‐ and cartilage‐derived morphogenetic proteins (BMPs and CDMPs), which are TGFβ superfamily members, are growth and differentiation factors that have been recently isolated, cloned and biologically characterized. They are important regulators of key events in the processes of bone formation during embryogenesis, postnatal growth, remodelling and regeneration of the skeleton. In the present study, we used immunohistochemical methods to investigate the distribution of BMP‐2, ‐3, ‐5, ‐6, ‐7 and CDMP‐1, ‐2, ‐3 in human osteophytes (abnormal bony outgrowths) isolated from osteoarthritic hip and knee joints from patients undergoing total joint replacement surgery. All osteophytes consisted of three different areas of active bone formation: (1) endochondral bone formation within cartilage residues; (2) intramembranous bone formation within the fibrous tissue cover and (3) bone formation within bone marrow spaces. The immunohistochemistry of certain BMPs and CDMPs in each of these three different bone formation sites was determined. The results indicate that each BMP has a distinct pattern of distribution. Immunoreactivity for BMP‐2 was observed in fibrous tissue matrix as well as in osteoblasts; BMP‐3 was mainly present in osteoblasts; BMP‐6 was restricted to young osteocytes and bone matrix; BMP‐7 was observed in hypertrophic chondrocytes, osteoblasts and young osteocytes of both endochondral and intramembranous bone formation sites. CDMP‐1, ‐2 and ‐3 were strongly expressed in all cartilage cells. Surprisingly, BMP‐3 and ‐6 were found in osteoclasts at the sites of bone resorption. Since a similar distribution pattern of bone morphogenetic proteins was observed during embryonal bone development, it is suggested that osteophyte formation is regulated by the same molecular mechanism as normal bone during embryogenesis.     

Self-support of induced bone tissue

Ability of osteogenic precursors of bone marrow from intact and induced (by implantation of decalcified bone matrix) bones to support themselves was compared by transplantation in diffusion chambers. Osteogenesis continued for several months in bone marrow transplants from intact bones whereas in transplants from induced bone tissue active osteogenesis, although still observed after 12–20 days, ceased after 2 months. Fibroblasts from the second passage from cultures of hematopoietic tissue of induced bones, unlike fibroblasts from cultures of intact bone marrow, were virtually without osteogenic powers. These results confirm that induced osteogenic tissue has limited ability of self-support after the action of the inducer has ceased, compared with intact bone tissue.N. F. Gamaleya Institute of Epidemiology and Microbiology, Moscow, (Presented by Academician of the Academy of Medical Sciences of the USSR O. V. Baroyan.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 81, No. 2, pp. 239–242, February, 1976.     

骨髓间充质干细胞与同种异体骨复合修复犬下颌骨缺损：成骨能力检测

 背景：同种异体骨与自体骨有相似解剖外形和生物学特性,是较佳的生物支架材料。自体骨髓来源的间充质干细胞具有多分化潜能,能向成骨、成软骨细胞分化,加速骨组织及软骨组织的形成。目的：探讨同种异体骨支架复合自体骨髓间充质干细胞促进犬下颌骨半侧缺损的新骨成骨能力。方法：拔出24只比格犬左侧下颌牙,伤口愈合后2个月,人为造成犬下颌骨缺损,对照组用单纯冻干同种异体骨修复,实验组用同种异体冻干骨加自体骨髓间充质干细胞修复。术后4,12,24周对下颌骨体部进行骨密度扫描以及Micro-CT检查。结果与结论：实验组移植后12周开始,下颌骨的骨密度显著高于对照组(P < 0.05),随着时间推移,实验组和对照组骨密度均增高,但实验组增高明显高于对照组。随时间推移,实验组骨结构参数成阶梯式递增或递减,对照组虽也有递增或递减,但不明显。术后24周实验组感兴趣区骨小梁分离度大于对照组(P < 0.05),骨体积分数、骨小梁数量、骨小梁厚度小于对照组(P < 0.05)。结果表明骨髓间充质干细胞能加速同种异体骨的骨改建速度。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：     

In vitro osteogenesis assays: influence of the primary cell source on alkaline phosphatase activity and mineralization

In trabecular bone fracture repair in vivo, osteogenesis occurs through endochondral ossification under hypoxic conditions, or through woven bone deposition in the vicinity of blood vessels. In vitro osteogenesis assays are routinely used to test osteoblastic responses to drugs, hormones, and biomaterials for bone and cartilage repair applications. These cell culture models recapitulate events that occur in woven bone synthesis, and are carried out using primary osteoblasts, osteoblast precursors such as bone marrow-derived mesenchymal stromal cells (BMSCs), or various osteoblast cell lines. With time in culture, cell differentiation is typically assessed by examining levels of alkaline phosphatase activity (an early osteoblast marker) and by evaluating the assembly of a collagen (type I)-containing fibrillar extracellular matrix that mineralizes. In this review, we have made a comparative analysis of published osteogenic assays using calvarial cells, calvaria-derived cell lines, and bone marrow stromal cells. In all of these cell types, alkaline phosphatase activity shows similar progression over time using a variety of osteogenic and mineralizing media conditions; however, levels of alkaline phosphatase activity are not proportional to observed mineralization levels.     

Alveolar mononuclear cells can develop into multinucleated osteoclasts: an in vitro cell culture model

Previous studies have shown that osteoclasts are derived from mononuclear cells of hemopoietic bone marrow and peripheral blood. The purpose of this study was to demonstrate the presence of multinucleated osteoclasts after adding alveolar mononuclear cells to new-born rat calvaria osteoblasts in vitro. To utilize osteoclast-free bone, fetal calvariae were obtained from newborn Wistar-rats and cultured in DMEM medium for 14 days. On the day of osteoblast culture, alveolar mononuclear cells were isolated from newborn Wistar rats with a serial washing method and then co-cultured with the calvarial osteoblasts. Bone resorption characteristics were observed both with light and scanning electron microscopy. When alveolar mononuclear cells were cultured for 14 days on the calvarial osteoblasts in response to 1 alpha, 25-dihydroxyvitamin D3, they formed tartrate-resistant acid phosphatase (TRAP)-positive mononuclear and multinucleated cells. Resorption pits were seen in the 7-14 days long-term cultures. These results indicate that osteoclasts can be derived from alveolar mononuclear cells in vitro when a suitable microenvironment is provided by calvarial osteoblasts and vitamin D(3).     

膝关节腔内培养骨髓间充质干细胞复合脱钙骨的组织工程软骨

背景：如何更好地以组织工程学方法修复关节软骨缺损并达到良好的远期疗效目前尚无公识。 目的：创新性地在膝关节腔内培养兔骨髓间充质干细胞复合同种异体脱钙骨的组织工程软骨。  方法：采用全骨髓贴壁筛选法分离培养兔骨髓间充质干细胞,DMEM/F12完全培养基培养,成软骨诱导条件培养基诱导分化。取同种异体兔的髂骨和椎体骨制作成脱钙骨支架,诱导后的骨髓间充质干细胞种植于脱钙骨支架上,培养1 d后将细胞-支架复合物用筋膜包裹置于兔左膝关节腔内培养,单纯脱钙骨支架筋膜包裹置入右膝关节腔。于培养第4,8,12周分别取材,行大体观察并制成石蜡切片,采用苏木精-伊红染色、甲苯胺蓝染色,Ⅱ型胶原免疫组化染色方法进行组织学观察。 结果与结论：培养4,8周,细胞-支架组标本Ⅱ型胶原免疫组化的平均吸光度值(A)分别为0.263±0.031,0.340±0.052,单纯支架组标本分别为0.147±0.027,0.165±0.030,两组比较差异有显著性意义(P < 0.05);培养12周细胞-支架组标本Ⅱ型胶原免疫组化A值平均为0.362±0.037,标本类似正常软骨外观,Ⅱ型胶原免疫组化反应呈阳性;而单纯支架组脱钙骨支架降解。培养12周细胞-支架组苏木精-伊红染色结果显示细胞数量多,脱钙骨支架基本被吸收;而甲苯胺蓝染色结果显示有被染成紫红色的异染性基质形成。结果提示兔骨髓间充质干细胞复合同种异体脱钙骨可在兔膝关节腔内培养出组织工程软骨。     

Repair of calvarial defects with customised tissue-engineered bone grafts II. Evaluation of cellular efficiency and efficacy in vivo

We have demonstrated in Part I of this study [see Schantz, J.-T., et al., Tissue Eng. 2003;9(Suppl. 1): S-113-S-126; this issue] that bone marrow-derived progenitor cells and calvarial osteoblasts could be successfully directed into the osteogenic lineage and cultured in three-dimensional (3-D) polycaprolactone (PCL) scaffolds. The objective of the second part of the study was to evaluate and to compare tissue engineered cell-polymer constructs using calvarial osteoblasts (group I) and mesenchymal progenitor cells (MPCs; group II) for the reconstruction of critical-size and three-dimensionally complex cranial defects. In 30 New Zealand White rabbits, bilateral parietal critical-size defects were created. On the basis of computed tomography scans, customized PCL scaffolds with precisely controlled microarchitecture were fabricated, using a rapid prototyping technology. Bone marrow-derived progenitor cells and osteoblasts were isolated and expanded in culture. Osteoblasts (group I) and mesenchymal progenitor cells (group II) were seeded in combination with a fibrin glue suspension into 40 PCL scaffolds. After incubating for 3 days in static culture, the PCL scaffold-cell constructs as well as nonseeded PCL scaffolds (control group) were implanted into 15-mm-diameter calvarial defects. Reconstruction of the cranium and bone formation were evaluated after 3 months. In vivo results indicated osseous tissue integration within the implant and functionally stable restoration of the calvarium. Islands of early bone formation could be observed in X-ray radiographs and in histological sections. Implants showed a high cell:ECM ratio and a dense vascular network. Mechanical testing of the reconstructed area revealed partial integration with the surrounding corticocancellous calvarial bone. The amount (area) of calcification, measured by clinical computed tomography, indicated that cell-seeded constructs measured about 60% more than unrepaired or unseeded scaffolds. Mechanical investigations revealed that stiffness reached 52 +/- 29 and 44 +/- 16 MPa for MPC- and osteoblast-seeded scaffolds, respectively. The yield strength for the push-out tests was 180 +/- 36 N for normal calvarial bone, 90 +/- 1 N for unrepaired site, and 106 +/- 10 N for unseeded constructs, which is about 60% of normal bone strength. MPC- and osteoblast-seeded scaffolds indicated a yield strength of 149 +/- 15 and 164 +/- 42 N, respectively, which is about 85-90% of normal bone. This study demonstrated that customized biodegradable polymeric implants may be used to deliver osteogenic cells and enhance bone formation within critically-sized defects in vivo. The use of rapid prototyping technology to produce scaffolds with controlled external geometry and microarchitecture offers new possibilities in the functional and aesthetic reconstruction of complex craniofacial defects.     

Histological comparison of bone induced from autogenously grafted periosteum with bone induced from autogenously grafted bone marrow in the rat calvarial defect model

Both periosteum and bone marrow have the potential to induce heterotopic bone when grafted. Whether the process of bone formation is controlled by the recipient environment where the donor graft is placed or by factors from the donor site is not well documented. The purpose of this study was to examine the histology of new bone induced by either autogenously grafted periosteum or autogenously grafted bone marrow using the rat calvarial defect model in Sprague–Dawley rats. Grafts of either bone marrow or periosteum obtained from tibias were placed in calvarial defects with beta-tricalcium phosphate. Ten days after grafting, active cell proliferation was observed in the defects of both types of grafts. After 20 days, cancellous bone formation was observed in the defects with bone marrow grafts, and intramembranous bone formation was observed in the defects with periosteal grafts. After 30 days, bone marrow grafts had developed bone with a bone marrow-like structure, and the periosteal grafts had produced cortical bone structure in the defects. The findings suggest that the type of bone formation is determined by characteristics of the donor site.     

An autoradiographic study of chondrocyte transformation into chondroclasts and osteocytes during bone formation In vitro

Excised mouse pubic bone rudiments were exposed to H³-thymidine. Rudiments preserved immediately after exposure consisted of mesenchyme with a large number of cells showing intense radioactivity. Rudiments incubated on a filter membrane after exposure went through the developmental stages of complete chondrification of the pubic rami followed by periosteal and then endochondral bone formation. Only chondrocytes showed radioactivity in rami consisting of cartilage and periosteal bone that were preserved prior to endochondral ossification. Cell types showing radioactivity in rami preserved during endochondral ossification were chondrocytes, chondroclasts, and osteoblasts and osteocytes of endochondral bone. The results of the study demonstrated that hypertrophic chondrocytes of the calcified cartilage of a developing mammalian long bone not only survive dissolution of their matrix, but transform into chondroclasts and osteoprogenitor cells that give rise to osteoblasts and osteocytes which form endochondral bone in the absence of blood vessels.     

基因修饰骨髓间充质干细胞修复关节软骨损伤

背景：随着生物技术的发展,通过转基因技术修饰细胞,从而获得长期稳定表达的生物活性因子以治疗关节软骨损伤逐渐引起重视。 目的：就基因修饰的骨髓间充质干细胞在修复关节软骨损伤中的应用作一综述。 方法：由第一作者检索1990至2011年PubMed数据库(http://www.ncbi.nlm.nih.gov/PubMed)有关基因修饰骨髓间充质干细胞修复关节软骨损伤的文献,英文检索词为“cartilage,gene therapy,mesenchymal stem cells,tissue engineering,bioactive factor,vector”。共纳入15篇文献归纳总结。 结果与结论：骨髓间充质干细胞已被广泛应用于修复关节软骨损伤。通过转基因技术将特定外源基因导入骨髓间充质干细胞,联合细胞治疗和基因治疗可达到更好的治疗效果,在关节软骨损伤的治疗中有广阔的应用前景。     

Morphological influence of ascorbic acid deficiency on endochondral ossification in osteogenic disorder Shionogi rat

The influences of chronic deficiency of L-ascorbic acid (AsA) on the differentiation of osteo-chondrogenic cells and the process of endochondral ossification were examined in the mandibular condyle and the tibial epiphysis and metaphysis by using Osteogenic Disorder Shionogi (ODS) rats that bear an inborn deficiency of L-gulonolactone oxidase. Weanling male rats were kept on an AsA-free diet for up to 4 weeks, until the symptoms of scurvy became evident. The tibiae and condylar processes of scorbutic rats displayed undersized and distorted profiles with thin cortical and scanty cancellous bones. In these scorbutic bones, the osteoblasts showed characteristic expanded round profiles of rough endoplasmic reticulum, and lay on the bone surface where the osteoid layer was missing. Trabeculae formation was deadlocked, although calcification of the cartilage matrix proceeded in both types of bone. Scorbutic condylar cartilage showed severe disorganization of cell zones, such as unusual thickening of the calcification zone, whereas the tibial cartilage showed no particular alterations (except for a moderately decreased population of chondrocytes). In condylar cartilage, hypertrophic chondrocytes were encased in a thickened calcification zone, and groups of nonhypertrophic chondrocytes occasionally formed cell nests surrounded by a metachromatic matrix in the hypertrophic cell zone. These results indicate that during endochondral ossification, chronic AsA deficiency depresses osteoblast function and disturbs the differentiation pathway of chondrocytes. The influence of scurvy on mandibular condyle cartilage is different from that on articular and epiphyseal cartilage of the tibia, suggesting that AsA plays different roles in endochondral ossification in the mandibular condyle and long bones.     

组织工程化骨修复犬牙槽骨缺损的组织学观察

目的观察组织工程化骨修复犬牙槽骨缺损过程中的新骨形成及其矿化程度。方法全麻及无菌条件下抽取犬胸骨骨髓,体外诱导培养犬自体骨髓间充质干细胞。取成年杂种犬8只,随机分成实验组和对照组,每组4只。实验组将已分化的自体成骨细胞与Bio-Oss骨胶原复合修复犬牙槽骨缺损:对照组牙槽骨缺损处仅植入Bio-Oss骨胶原。每组分别于术后4周,8周各处死2只动物,标本常规切片后行HE和改良Masson三色染色,光学显微镜下观察各组标本的组织学表现。结果实验组4周时即可见骨缺损修复区骨胶原内蓝色的新骨形成,随着时间的推移,形成新骨逐渐矿化成为红色的成熟骨组织,8周时骨缺损修复区可见大片新骨呈岛状或条索状排列,新生牙槽骨骨化效果明显优于对照组,Masson染色为红色。结论组织工程化骨促进犬牙槽骨缺损组织的修复。     

In vivo bone generation via the endochondral pathway on three-dimensional electrospun fibers

A new concept of generating bone tissue via the endochondral route might be superior to the standard intramembranous ossification approach. To implement the endochondral approach, suitable scaffolds are required to provide a three-dimensional (3-D) substrate for cell population and differentiation, and eventually for the generation of osteochondral tissue. Therefore, a novel wet-electrospinning system, using ethanol as the collecting medium, was exploited in this study to fabricate a cotton-like poly(lactic-co-glycolic acid)/poly(ε-caprolactone) scaffold that consisted of a very loose and uncompressed accumulation of fibers. Rat bone marrow cells were seeded on these scaffolds and chondrogenically differentiated in vitro for 4 weeks followed by subcutaneous implantation in vivo for 8 weeks. Cell pellets were used as a control. A glycosaminoglycan assay and Safranin O staining showed that the cells infiltrated throughout the scaffolds and deposited an abundant cartilage matrix after in vitro chondrogenic priming. Histological analysis of the in vivo samples revealed extensive new bone formation through the remodeling of the cartilage template. In conclusion, using the wet-electrospinning method, we are able to create a 3-D scaffold in which bone tissue can be formed via the endochondral pathway. This system can be easily processed for various assays and histological analysis. Consequently, it is more efficient than the traditional cell pellets as a tool to study endochondral bone formation for tissue engineering purposes.     

In vivo evaluation of a porous hydroxyapatite/poly-DL-lactide composite for bone tissue engineering

As reported previously, a porous composite of uncalcined hydroxyapatite (u-HA) and poly-DL-lactide (PDLLA) showed excellent osteoconductivity and biodegradability as a bone substitute in rabbit model. In this study, to investigate the usefulness of this composite as a scaffold loaded with cells, we estimated whether this material showed osteogenesis on implantation to extraosseous site. On loading with syngeneic bone marrow cells and implantation into rat dorsal subcutaneous tissue, osteogenesis with enchondral ossification was seen both on and in the material at 3 weeks after implantation. The osteogenesis in the u-HA/PDLLA had progressed, and newly formed bone tissue was found in the material by 6 weeks. To investigate the osteoinductive properties of the material, we implanted this porous composite material into extraosseous canine dorsal muscle. At 8 weeks, osteogenesis was seen in the pores of the material. Newly formed bone could be observed adjacent to the material. In addition, cuboidal osteoblasts adjacent to the newly formed bone were evident. Neither cartilage nor chondrocytes were found. These results might indicate that the material induced osteogenesis by intramembranous ossification. Conversely, similar porous PDLLA did not induce osteogenesis during the observation period. Therefore, porous HA/PDLLA, which has osteoconductive and osteoinductive properties, might be a useful material for use as a bone substitute and cellular scaffold.     

Structure, formation and role of cartilage canals in the developing bone

In the long bones, endochondral bone formation proceeds via the development of a diaphyseal primary ossification centre (POC) and an epiphyseal secondary ossification centre (SOC). The growth plate, the essential structure for longitudinal bone growth, is located between these two sites of ossification. Basically, endochondral bone development depends upon neovascularization, and the early generation of vascularized cartilage canals is an initial event, clearly preceding the formation of the SOC. These canals form a discrete network within the cartilaginous epiphysis giving rise to the formation of the marrow space followed by the establishment of the SOC. These processes require excavation of the provisional cartilaginous matrix which is eventually replaced by permanent bone matrix. In this review, we discuss the formation of the cartilage canals and the importance of their cells in the ossification process. Special attention is paid to the enzymes required in disintegration of the cartilaginous matrix which, in turn, will allow for the invasion of new vessels. Furthermore, we show that the mesenchymal cells of the cartilage canals express bone-relevant proteins and transform into osteocytes. We conclude that the canals are essential for normal epiphyseal bone development, the establishment of the growth plate and ultimately longitudinal growth of the bones.