Structural differences between bone formed intramuscularly following the transplantation of isolated calvarial bone cells or chondrocytes

Summary Bone formed in intramuscular transplants of isolated syngeneic calvarial bone cells in mice, was compared with endochondral bone induced by cartilage produced by analogous transplants of isolated epiphyseal chondrocytes, as well as with parietal bones forming the bulk of the calvaria. Transplanted calvarial cells produced islands of bone, some of which contained intraosseous cavities. Osteoclasts inside these cavities were observed only in 14-day-old transplants and bone marrow cells in 28-day and older transplants. On the contrary, bone marrow appeared soon after formation of bone trabeculae in endochondral bone. The percentage area occupied by bone marrow in these specimens was about twentyfold larger than in the bone formed by transplanted bone cells. On the other hand, the bone marrow area in the latter type of bone was somewhat smaller but of similar order as in parietal bones. Moreover, both in parietal bones and in bone formed by isolated bone cells, the bone marrow was devoid of fat cells which were numerous in bone arising by endochondral ossification. It appears, therefore, that the ratio of bone marrow to the bone tissue area in parietal bones depends more on the intrinsic properties of osteoblasts than on the local factors in the environment of the developing bone. In the case of bone induced by cartilage, the bone marrow/bone tissue area could be determined both by the extent of cartilage resorption by vascularized tissue and by the properties of osteoblasts.     

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).     

Canine cranial reconstruction using autologous bone marrow stromal cells

Limited-sized transplants of culture-expanded autologous or allogeneic bone marrow stromal cells (BMSCs) form cortico-cancellous bone in rodent models. Initiation of clinical studies using autologous BMSC transplantation requires effective bone formation among sizable transplants in a large animal model as well as noninvasive techniques for evaluating transplant success. Here, we obtained bone marrow from the femurs of six dogs and expanded BMSCs in tissue culture. Autologous BMSC-hydroxyapatite/tricalcium phosphate (HA/TCP) transplants were introduced into critical-sized calvarial defects and contralateral control skull defects received HA/TCP vehicle alone. At intervals ranging from 2 to 20 months, transplants were biopsied or harvested for histological and mechanical analysis. Noninvasive studies, including quantitative computed tomography scans and ultrasound, were simultaneously obtained. In all animals, BMSC-containing transplants formed significantly more bone than their control counterparts. BMSC-associated bone possessed mechanical properties similar to the adjacent normal bone, confirmed by both ultrasound and ex vivo analysis. Evaluation by quantitative computed tomography confirmed that the extent of bone formation demonstrated by histology could be discerned through noninvasive means. These results show that autologous cultured BMSC transplantation is a feasible therapy in clinical-sized bone defects and that such transplants can be assessed noninvasively, suggesting that this technique has potential for use in patients with certain bone defects.     

Experimental aspects of transplantation of haemopoietic cells of fetal liver

About 25% mice treated for radiation-induced bone marrow aplasia with allogeneic transplantation of histoincompatible fetal liver cells survived four months. The same survival time was observed in 80% of mice after transplantation of syngeneic fetal liver cells. Mice irradiated and not treated and those receiving transplants of allogeneic histoincompatible bone marrow died within a few weeks after transplantation. This observation confirms the reports on the therapeutic superiority of transplantation of haemopoietic fetal liver cells over transplantation of bone marrow of similar tissue incompatibility and reports on the usefulness of tissue matching, if possible. Another observation was an absence of inactivation of the haemopoietic stem cells in mixtures of genotypically different fetal liver cells. The animals receiving these cell mixtures showed survival rates similar to those of the animals receiving genetically homogenous cells. This opens theoretical possibilities for undertaking clinical trials of transplantation of cells from several fetal livers, especially in view of the accumulating reports that one human fetal liver may be too small to serve as a source of stem cells for ensuring successful transplantation.     

Osteoblast and osteoclast precursors in primary cultures of calvarial bone cells

Bone cells obtained by digestion of fetal mouse or chicken calvaria were tested for their ability to form or resorb bone in vitro. The isolated cells were precultured for 6 days and subsequently cocultured for 11 days with periosteum-free noninvaded fetal mouse long bone rudiments. Bone formation and resorption during coculture were evaluated by histology and 45Ca release from prelabeled bones. The calvarial origin of cells in cocultures was traced by labeling the cells with 3H-thymidine before coculture, followed by autoradiography. Many osteoblasts and osteoclasts as well as fibroblasts developed from mouse periosteal cells released late in the sequential digestion procedure and previously denoted as "osteoblastlike" (BL). No or few osteoblasts and osteoclasts but many fibroblasts developed from early released cell fractions that have previously been denoted as "osteoclastlike" (CL). Only osteoblasts and fibroblasts but not osteoclasts developed from chicken calvarial cell fractions. The osteoblasts developed primarily from cell fractions from the inner layer of the periosteum, previously denoted as "osteoblastlike" (OB). Cells obtained from the outer layer of the periosteum (PF) gave rise mainly to fibroblasts. These studies show that osteoblast and osteoclast precursor cells are maintained in monolayer cultures of periosteal cell fractions. However, sequential digestion of mouse calvaria does not lead to separation of the two types of bone cells. Rather, osteoclast and osteoblast precursors are released jointly, from the periosteal cell layers closest to the bone surface. In the chicken cell fractions osteoclast precursors are absent after preculture, resulting in a more homogeneous population of osteoblast and fibroblast but not osteoclast precursors.     

Bone grafts cultured with bone marrow stromal cells for the repair of critical bone defects: an experimental study in mice

Tissue engineering of autologous bone combined with osteoprogenitor cells is a suitable strategy for filling large bone defects. The aim of this study was to evaluate the osteogenicity of a xenogenic bone graft cultured with allogenic bone marrow stromal cells (BMSC) in a mouse critical size craniotomy. Bovine trabecular bone grafts were made free of bone marrow cells or debris and were delipidated. BMSC were harvested from C57BL/6-Tg(ACTbEGFP)1Osb/J mice (GFP+ cells) and were cultured 14 days on bone grafts in control or osteogenic medium. Engineered grafts were implanted in calvarial defect in C57BL/6 mice. Four groups were studied: graft with BMSC differentiated in osteoblasts (G-Ob), graft with BMSC (G-BMSC), graft without cells (G) and no graft. Calvariae were studied 2 and 8 weeks after implantation by radiographic and histomorphometric analyses. G group: the bone ingrowth was limited to the edges of the defect. The center of the graft was filled by a fibrovascular connective tissue. G-BMSC or G-Ob groups: bone formation occurred early in the center of the defect and did not increase between 2 and 8 weeks; the newly formed woven bone was partially replaced by lamellar bone. The preoperative osteoblastic differentiation of BMSC did not allow faster and better bone regeneration. After 2 weeks, GFP+ cells were observed around the grafted bone but no GFP+ osteocyte was present in the newly formed bone. No GFP+ cell was noted after 8 weeks. However, pre-implantation culture of the biomaterial with allogenic BMSC greatly enhanced the bone regeneration.     

同种异体骨髓间充质干细胞的研究现状

背景：自体骨髓间充质干细胞移植是近年来在治疗关节软骨缺损较新的方法,但在临床上,急性骨软骨缺损很难在短时间内应用自体骨髓间充质干细胞修复,而同种异体的骨髓间充质干细胞可以提前制备,以备应用。 目的：系统分析同种异体骨髓间充质干细胞的免疫调节及抑制研究。 方法：以“Bone Mesenchymal Stem Cells,immunomodulation activity,immunosuppression;同种异体骨髓基质干细胞,免疫调节活性,免疫抑制”为关键词,由第一作者检索1990/2009 PubMed及万方数据库有关同种异体骨髓间充质干细胞免疫调节及抑制研究方面的文献。 结果与结论：虽然移植同种异体细胞有可能被受体体内的免疫系统攻击,但骨髓间充质干细胞是例外,因为它是免疫调节细胞,具有抑制免疫反应的作用。骨髓间充质干细胞对T细胞、B细胞、自然杀伤细胞和抗原提呈细胞均具有抑制作用。骨髓间充质干细胞是从骨髓中分离出来的组织工程细胞群,它携有免疫抑制相关标志,并在体外抑制同种异体T细胞增殖,因此骨髓间充质干细胞可用于同种异体细胞治疗。骨髓间充质干细胞为临床上同种异体骨髓间充质干细胞修复于关节软骨缺损提供了一种崭新的方法。     

大鼠成骨细胞的原代培养和鉴定

背景：组织工程需要大量的种子细胞,成骨细胞已成为骨组织工程构建的重要种子细胞之一。但成骨细胞取材困难,获得的成骨细胞的纯度不一。 目的：建立大鼠新生乳鼠颅骨来源成骨细胞的分离培养纯化方法,观察颅骨来源成骨细胞生物学特点。 方法：采用二次酶消化法对SD大鼠乳鼠成骨细胞进行原代培养,扩增。通过差速贴壁法进行成骨细胞纯化。通过形态学、细胞碱性磷酸酶检测、茜素红染色、钙结节Von kossa法染色、超微结构以及细胞增殖曲线,确定其增殖与成骨活性。 结果与结论：二次酶消化法培养颅骨来源成骨细胞可获得原代细胞增殖,传代扩增细胞具有典型成骨细胞形态学和生物学活性。碱性磷酸酶、茜素红染色、钙结节Von kossa法染色均呈阳性结果。超微结构显示为高分化功能活跃成骨细胞,细胞增殖曲线显示细胞生长活跃。提示,新生SD大鼠颅骨来源成骨细胞具有良好的增殖与成骨活性,能连续传代增殖,纯度高,细胞生物学特征稳定,适用于做体外实验研究。     

Repair of calvarial defects with customized tissue-engineered bone grafts I. Evaluation of osteogenesis in a three-dimensional culture system

Bone generation by autogenous cell transplantation in combination with a biodegradable scaffold is one of the most promising techniques being developed in craniofacial surgery. The objective of this combined in vitro and in vivo study was to evaluate the morphology and osteogenic differentiation of bone marrow derived mesenchymal progenitor cells and calvarial osteoblasts in a two-dimensional (2-D) and three-dimensional (3-D) culture environment (Part I of this study) and their potential in combination with a biodegradable scaffold to reconstruct critical-size calvarial defects in an autologous animal model [Part II of this study; see Schantz, J.T., et al. Tissue Eng. 2003;9(Suppl. 1):S-127-S-139; this issue]. New Zealand White rabbits were used to isolate osteoblasts from calvarial bone chips and bone marrow stromal cells from iliac crest bone marrow aspirates. Multilineage differentiation potential was evaluated in a 2-D culture setting. After amplification, the cells were seeded within a fibrin matrix into a 3-D polycaprolactone (PCL) scaffold system. The constructs were cultured for up to 3 weeks in vitro and assayed for cell attachment and proliferation using phase-contrast light, confocal laser, and scanning electron microscopy and the MTS cell metabolic assay. Osteogenic differentiation was analyzed by determining the expression of alkaline phosphatase (ALP) and osteocalcin. The bone marrow-derived progenitor cells demonstrated the potential to be induced to the osteogenic, adipogenic, and chondrogenic pathways. In a 3-D environment, cell-seeded PCL scaffolds evaluated by confocal laser microscopy revealed continuous cell proliferation and homogeneous cell distribution within the PCL scaffolds. On osteogenic induction mesenchymal progenitor cells (12 U/L) produce significantly higher (p < 0.05) ALP activity than do osteoblasts (2 U/L); however, no significant differences were found in osteocalcin expression. In conclusion, this study showed that the combination of a mechanically stable synthetic framework (PCL scaffolds) and a biomimetic hydrogel (fibrin glue) provides a potential matrix for bone tissue-engineering applications. Comparison of osteogenic differentiation between the two mesenchymal cell sources revealed a similar pattern.     

Circulating thymic hormone levels in severe combined immunodeficiency.

Twenty-three patients with severe combined immunodeficiency disease were studied for circulating thymic hormone levels (facteur thymique serique, FTS), 21 prior to treatment by transplantation of bone marrow, thymus or fetal liver. Thirteen showed undetectable FTS activity. Only two had normal levels of this hormone. In serial determinations of FTS activity prior to and after transplantation, patients given bone marrow transplants developed sustained increments of serum FTS activity early in the course of their immunological reconstitution. However, patients given transplants of fetal liver alone or fetal liver plus thymus from fetuses of less than 12 weeks gestation generally did not show an increment of FTS activity during the period of observation. Transplantation of irradiated thymus derived from fetuses of more than 14 weeks gestation produced sustained increases of thymic hormone activity. These observations suggest that a cell of haematopoietic origin provides a stimulus necessary for differentiation or maturation of thymic secretory activity and that this cell(s) is present in post-natal marrow, but is either undeveloped or immature in the early fetal liver or fails to migrate to the thymus of an allogeneic host.     

Reduced Bone Resorption In Toothless (Osteopetrotic) Rats–An Abnormality of Osteoblasts Related to Their Inability to Activate Osteoclast Activity In Vitro

Osteopetrosis is a heterogeneous group of metabolic bone disorders characterized by reduced bone resorption. In the toothless (tl) osteopetrotic rat mutation there are few osteoclasts and mutants are not cured by bone marrow transplants. This suggests that the defect(s) in tl rats is within the skeletal microenvironment and not one of stem cell incompetence. Osteoblasts are known to play a role in bone resorption and abnormalities in these cells have been reported in tl rats. We explored the ability of osteoblasts from tl rats to activate resorption by normal osteoclasts when co-cultured in the presence of 1, 25-dihydroxyvitamin D (l, 25(OH)2D). Stimulation with l, 25(OH)2D produced a highly significant response in normal osteoblast co-cultures, but no response was observed in mutant cultures over a wide dose range. Ligand-binding studies demonstrated no abnormalities in vitamin D receptor (VDR) affinity, but mutant osteoblasts had reduced VDR numbers. Taken together with the demonstrated resistance of these mutants to the hypercalcemic effects of l, 25(OH)2D and parathyroid hormone in vivo, these data implicate osteoblasts in the pathogenesis of this mutation.     

Allogeneic Th1 Cells Home to Host Bone Marrow and Spleen and Mediate IFNγ-Dependent Aplasia

Bone marrow graft failure and poor graft function are frequent complications after hematopoietic stem cell transplantation and result in significant morbidity and mortality. Both conditions are associated with graft-versus-host disease (GVHD), although the mechanism remains undefined. Here we show, in 2 distinct murine models of GVHD (complete MHC- and class II-disparate) that mimic human peripheral blood stem cell transplantation, that Th1 CD4⁺ cells induce bone marrow failure in allogeneic recipients. Bone marrow failure after transplantation of allogeneic naïve CD4⁺ T cells was associated with increased CD4⁺ Th1 cell development within bone marrow and lymphoid tissues. Using IFNγ-reporter mice, we found that Th1 cells generated during GVHD induced bone marrow failure after transfers into secondary recipients. Homing studies demonstrated that transferred Th1 cells express CXCR4, which was associated with accumulation within bone marrow and spleen. Allogeneic Th1 cells were activated by radiation-resistant host bone marrow cells and induced bone marrow failure through an IFNγ-dependent mechanism. Thus, allogeneic Th1 CD4⁺ cells generated during GVHD traffic to hematopoietic sites and induce bone marrow failure via IFNγ-mediated toxicity. These results have important implications for prevention and treatment of bone marrow graft failure after hematopoietic stem cell transplantation.     

The thymus in patients with allogeneic bone marrow transplants.

The thymus glands from 11 patients with aplastic anemia or acute leukemia who received allogeneic bone marrow transplants were studied at autopsy. All showed marked cortical involution. In the short-term survivors the medulla and perivascular spaces were lymphocyte-depleted and the epithelial cells formed pseudorosettes. In those surviving over 2 months, increasing numbers of small lymphocytes were present, presumably reconstituted with donor lymphocytes. Phagocytosis of cellular debris was frequent, especially in patients with graft-versus-host reaction (GVHR) or treated with anithymocyte globulin (ATG). Plasma cells were numerous in perilobular tissue and were occasionally found within the medulla. The findings are compatible with the concept that the thymus plays an important role in the immune deficiency experienced after allogeneic bone marrow transplantation and in the subsequent lymphoid reconstitution.     

Clonal hematopoiesis demonstrated by X-linked DNA polymorphisms after allogeneic bone marrow transplantation

We analyzed DNA from peripheral-blood and marrow cells from 12 recipients of allogeneic bone marrow transplants to determine whether monoclonal but otherwise normal hematopoiesis occurs in such patients. All patients were being treated for various forms of leukemia or lymphoma. In 10 patients, granulocytes isolated from peripheral-blood samples obtained 28 to 159 days after transplantation were polyclonal. In some, circulating T cells were isolated and also found to be polyclonal. In contrast, two patients had donor-derived monoclonal or oligoclonal hematopoiesis after transplantation. In one, DNA from circulating mononuclear cells obtained 29 days after transplantation revealed a monoclonal pattern on analysis of a restriction-fragment-length polymorphism in the phosphoglycerate kinase gene. In the other, analysis of a restriction-fragment-length polymorphism in the hypoxanthine phosphoribosyltransferase gene suggested the presence of a dominant clone in the granulocytes sampled 36 days after transplantation. When the latter patient was reassessed on day 267, the same clone of donor hematopoietic cells was still predominant and was found to include circulating T cells as well as granulocytes. We conclude that monoclonal hematopoiesis of donor origin may be observed in recipients of allogeneic bone marrow transplants, indicating that stem cells in normal adult human marrow are able to repopulate both lymphoid and myeloid compartments after transplantation.     

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。     

Listeriosis in Recipients of Allogeneic Bone Marrow Transplants from Unrelated Donors

 Two cases of listeriosis in patients submitted to matched unrelated donor bone marrow transplantation are reported. The patients developed listerial septicemia and listerial septicemia with meningitis and encephalitis 39 and 29 days after transplantation, respectively. Including the present two cases, 19 Listeria monocytogenes infections in related and unrelated donor allogeneic bone marrow transplant recipients have been reported to date. Infection occurred earlier in unrelated donor transplant recipients. Listeriosis is a rare complication in allogeneic bone marrow transplant recipients; however, the widespread practice of performing transplants from a donor-alternative to a human leukocyte antigen-compatible sibling and, in this setting, the need for intensified immunosuppression may predict an increasing and earlier occurrence of listeriosis.     

Allogeneic bone marrow transplantation in multiple myeloma. European Group for Bone Marrow Transplantation

BACKGROUND AND METHODS. In contrast to autologous bone marrow transplants for hematologic cancers, allogeneic transplants contain no tumor cells that might cause a relapse. We report the results of such allogeneic bone marrow transplantation using HLA-compatible sibling donors in 90 patients with multiple myeloma performed in 26 European centers between 1983 and 1989. RESULTS. At the time of the most recent follow-up, 79 months after the start of the study, 47 patients were alive and 43 were dead. The rate of complete remission after bone marrow transplantation was 43 percent for all patients and 58 percent for the patients who had engraftment. The actuarial survival at 76 months was 40 percent. The median duration of relapse-free survival among patients who were in complete remission after bone marrow transplantation was 48 months. The stage of the disease at diagnosis and the number of treatment regimens tried before bone marrow transplantation were predictive of the likelihood of complete remission after engraftment. There were trends toward longer survival among patients who were responsive to treatment before bone marrow transplantation, patients with Stage I disease at diagnosis, and patients who had received only first-line treatment before transplantation, as compared with those who were not responsive, those with Stage II or III disease at diagnosis, and those who had received three or more lines of treatment, but the differences in these factors were not statistically significant. Two post-transplantation factors predicted better long-term survival: complete remission after engraftment and grade I graft-versus-host disease, rather than grade II, III, or IV. CONCLUSIONS. Allogeneic bone marrow transplantation with the use of HLA-matched sibling donors appears to be a promising method of treatment for some patients with multiple myeloma.     

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.