Osteogenic potential of allogeneic rat marrow cells in porous hydroxyapatite ceramics: A histological study

Hydroxyapatite ceramics facilitate osteogenesis but cannot induce bone formation by themselves. We studied the feasibility of bone formation supported by allogeneic bone marrow cells in porous hydroxyapatite ceramics. Coralline hydroxyapatite discs were soaked in a marrow cell suspension harvested from either ACI (RT1^a), Lewis (RT1¹), or Fischer 344 (RT1^1v) male rats, and these discs were implanted subcutaneously into 56 male Fischer 344 rats. FK-506 (tacrolimus hydrate), an immunosuppressant, or saline was injected intramuscularly into the recipients every day for 2 weeks after surgery, and additional injections were given to 19 of the rats every 2 days for 2 more weeks. Neither of the mismatched major (ACI rat) or minor (Lewis rat) marrow cell transplants showed any bone formation without administration of FK-506. However, in rats treated with FK-506, bone formed in the pores of all the three types of ceramics implanted, which each contained the marrow cells from one of the three kinds of rats used. There were no differences among the three groups of donors with regard to the bone formation ratio. We previously reported that subcutaneous implantation of porous hydroxyapatite combined with isogeneic marrow cells resulted in consistent bone formation, even at ectopic sites. Since it would be difficult to harvest a large number of autologous marrow cells in clinical cases, we attempted to use allogeneic marrow cells and have shown the allogeneic murine marrow cells to have osteogenic potential.     

Osteogenic capacity of rat and human marrow cells in porous ceramics. Experiments in athymic (nude) mice

Porous hydroxyapatite ceramics, alone and combined with rat marrow cells, were implanted subcutaneously in 22 nude mice. The ceramics alone were invaded by fibrovascular tissue without any bone formation. In contrast, all the ceramics combined with marrow cells had bone formation in the pores 4 to 8 weeks after implantation. The bone formation began on the surface of the ceramic with direct bonding of the bone to the ceramic and proceeded to the center of the pores. The ceramics were also combined with bone marrow cells from 7 humans and implanted in nude mice. In five experiments, bone formation occurred after implantation. In addition, the ceramics were combined with in vitro cultured fibroblastic cells, resulting in bone formation in 2/6 cases. Our results indicate that the osteogenic ability of human marrow cells is sustained by porous hydroxyapatite ceramics.     

Osteogenic capacity of rat and human marrow cells in porous ceramics: Experiments in athymic (nude) mice

Porous hydroxyapatite ceramics, alone and combined with rat marrow cells, were implanted subcutaneously in 22 nude mice. The ceramics alone were invaded by fibrovascular tissue without any bone formation. In contrast, all the ceramics combined with marrow cells had bone formation in the pores 4 to 8 weeks after implantation. The bone formation began on the surface of the ceramic with direct bonding of the bone to the ceramic and proceeded to the center of the pores.

The ceramics were also combined with bone marrow cells from 7 humans and implanted in nude mice. In five experiments, bone formation occurred after implantation. In addition, the ceramics were combined with in vitro cultured fibroblastic cells, resulting in bone formation in 2/6 cases. Our results indicate that the osteogenic ability of human marrow cells is sustained by porous hydroxyapatite ceramics.     

Heterotopic osteogenesis in porous ceramics induced by marrow cells

When untreated porous calcium phosphate ceramics were transplanted into subcutaneous (s.c.) or intramuscular (i.m.) sites, fibrovascular tissue grew in the pore region without evidence of bone formation. However, when these same ceramics were combined with syngeneic marrow cells, osteogenesis was observed inside the pore region of the implanted ceramic. The osteogenesis began on the surface of the pore region at approximately 3 weeks postimplantation by a process of intramembranous bone formation, with the de novo bone tissue observed directly interfacing with the ceramic surface. Infrequently, small isolated areas showed cartilage formation with no noticeable endochondral ossification. At 4 weeks postimplantation of the ceramic with marrow cells, the osteogenesis in the ceramic accompanied an observed increase in compressive strength, rigidity, and energy absorption of the ceramic. These results suggest that a combination of porous ceramics and marrow cells may be useful for clinical problems requiring osseous reconstruction.     

Bone tissue engineering using novel interconnected porous hydroxyapatite ceramics combined with marrow mesenchymal cells: quantitative and three-dimensional image analysis

We developed fully opened interconnected porous calcium hydroxyapatite ceramics having two different pore sizes. One has pores with an average size of 150 microm in diameter, an average 40-microm interconnecting pore diameter, and 75% porosity (HA150). The other has pores with an average size of 300 microm in diameter, an average 60-100-microm interconnecting pore diameter, and 75% porosity (HA300). Because of its smaller pore diameter, HA150 has greater mechanical strength than that of HA300. These ceramics were combined with rat marrow mesenchymal cells and cultured for 2 weeks in the presence of dexamethasone. The cultured ceramics were then implanted into subcutaneous sites in syngeneic rats and harvested 2-8 weeks after implantation. All the implants showed bone formation inside the pore areas as evidenced by decalcified histological sections and microcomputed tomography images, which enabled three-dimensional analysis of the newly formed bone and calculation of the bone volume in the implants. The bone volume increased over time. At 8 weeks after implantation, extensive bone volume was detected not only in the surface pore areas but also in the center pore areas of the implants. A high degree of alkaline phosphatase activity with a peak at 2 weeks and a high level of osteocalcin with a gradual increase over time were detected in the implants. The levels of these biochemical parameters were higher in HA150 than in HA300. The results indicate that a combination of HA150 and mesenchymal cells could be used as an excellent bone graft substitute because of its mechanical properties and capability of inducing bone formation.     

Non-specific inflammation and bone marrow depletion due to intramedullary porous hydroxyapatite application.

Calcium hydroxyapatite (HA) ceramics are widely used in orthopaedic surgery and are known to be biocompatible, biodegradable, and osteoinductive. Recent studies of HA have identified early stage inflammatory reactions and third-body wear following hip replacement surgery. The aim of this study was to investigate the early stage inflammatory response toward HA by means of conventional radiography, histopathology, and scanning electron microscopy (SEM). HA was implanted into the medullary area of the tibiae of rabbits and followed from one week to six months. Radiography revealed local osteoporosis on Week 6 and Month 6 in some, but not, all rabbits. SEM presented favorable bonding between the implant and the cortical bone. Eosinophilic deposition, non-specific local inflammation, and bone marrow depletion were the histopathological findings. Multinuclear cells were identified in the inflammation area. Although bone ingrowth into the pores of the HA was demonstrated, there is no doubt that HA may cause an early inflammatory reaction in the bone marrow. This inflammatory reaction is followed by bone marrow depletion and may be the early sign and the cause of HA-coated implant failure.     

Anterior cervical fusion using porous hydroxyapatite ceramics for cervical disc herniation. a two-year follow-up.

BACKGROUND CONTEXT: The Smith-Robinson Method (SR), which employs autogenous bone, is the current standard for anterior cervical fusion (AF) surgery. However, autogenous bone has graft-related complications and morbidity, and harvesting it increases trauma and risk to the patient. The use of hydroxyapatite ceramic (HAP) inserts may provide a superior alternative. PURPOSE: To determine the efficacy of using HAP in AF. STUDY DESIGN/SETTING: A retrospective study of patients who had AF surgery with wide decompression and porous HAP inserts used to treat cervical disc herniation (CHD). PATIENT SAMPLE: We evaluated 36 patients who had single-level AF using HAP for CHD, without internal fixations, clinically and radiographically with a minimum follow-up of 2 years. There were 25 men and 11 women, with an average age of 49 years (age range, 24-78 years). Preoperative diagnosis included 25 cases with myelopathy and 11 cases with radiculopathy. OUTCOME MEASURES: We established four grades to classify the degree of bony fusion between the HAP and vertebra, based on any motion at the fused segment, any radiolucent zones (RZ) between vertebral bodies and the grafted HAP, and anterior or posterior bone formations on grafted HAPs. We evaluated the severity of myelopathy by applying the Japan Orthopaedic Association (JOA) scoring system. We evaluated the surgical outcome of the myelopathy patients using the Hirabayashi recovery rating, and for the radiculopathy patients, we used the Herkowitz criteria. METHODS: We retrospectively reviewed the radiographic and clinical records of all 36 patients from surgery up to periods ranging from 2 to 7 years after surgery, with the average period of follow-up being 4.5 years. We systematically classified the degree of bony fusion into four grades ranging from Grade 1 nonunion to Grade 4 complete union. RESULTS: None of the subjects showed Grades 1 and 2 fusion. Eleven percent of the cases showed Grade 3 and 89% showed Grade 4. Loss of height of the fused segment was observed in 29 cases with an average of 1.6 mm. A decrease of lordotic angle of the fused segment was observed in six cases with an average of 2.3 degrees. Four cases revealed cracked HAP inserts but achieved Grade 4 bone fusion. There was no evidence of collapse or displacement of HAPs. The results of the 11 radiculopathy patients were excellent in 10 cases and good in the remaining case. The recovery rate of the 25 myelopathy patients was 73.0%. CONCLUSIONS: Our method of anterior cervical fusion surgery using porous HAP inserted into resected end plates, combined with a wide decompression procedure, had clinical and radiographic results so satisfactory that we conclude that it can effectively replace the use of autogenous bone for treating cervical disc herniation.     

Augmentation of tendon attachment to porous ceramics by bone marrow stromal cells in a rabbit model

Tendon attachment to interconnected porous calcium hydroxyapatite ceramics (IP-CHA) with cultured bone marrow stromal cells (BMSC) was analysed. The purpose of this study was to evaluate whether BMSC in IP-CHA could augment the tendon attachment to IP-CHA histologically and biomechanically. Eighteen Japanese white rabbits were used. Cultured BMSCs were subcultured in IP-CHA. The grafted tendon and IP-CHA with BMSC complex were implanted in a bone defect of the knee [BMSC(+) group]. In the contralateral knee, a tendon and IP-CHA without BMSC complex were implanted [BMSC(-) group]. Histological findings of the interface between the tendon and IP-CHA were similar in the two groups 3 weeks after the operation. However, 6 weeks after the operation, more abundant bone formation around the tendon was observed in the BMSC(+) group. The direct apposition of the tendon to bone in pores and collagen fibre continuity between the tendon and fibrous tissue in pores were observed. In biomechanical evaluation, the maximum pull-out load of the tendon from the IP-CHA in the BMSC(+) group was significantly higher than that in the BMSC(-) group 6 weeks after the operation. BMSCs cultured in IP-CHA could augment tendon attachment to IP-CHA.

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Résumé Le propos de cette étude est de déterminer si l’utilisation de culture cellulaire de moelle osseuse (BMSC) avec l’utilisation de calcium d’hydroxyapatite (IP-CHA) augmente la résistance de l’attachement osseux du tendon sur le plan histologique et biomécanique. Dix-huit lapins blancs du Japon ont été utilisés pour ce travail. Les tendons greffés avec hydroxyapatite et culture de la cellule de moelle osseuse ont été implantés dans une defect au niveau du genou. Sur le genou opposé, le tendon a été implanté sans culture cellulaire. Sur le plan histologique les résultats sont identiques dans les deux groupes, 3 semaines après intervention, cependant 6 semaines après l’intervention la progression de la formation osseuse est plus importante autour du tendon ayant bénéficié d’une culture cellulaire de moelle osseuse. Il en est de même en ce qui concerne la résistance biomécanique du tendon qui est significativement plus importante dans le groupe culture cellulaire, 6 semaines après l’intervention.

     

大鼠骨髓间充质干细胞的分离、培养及表型鉴定

目的 建立大鼠骨髓间充质干细胞分离及培养的方法,探讨体外培养骨髓间充质干细胞的生物学特性.方法 采用密度梯度离心法结合贴壁筛选法分离纯化大鼠骨髓间充质干细胞,传代扩增,测定生长曲线,镜下连续观察细胞的形态变化.流式细胞仪鉴定其表面抗原 CD29、CD34、CD44和CD45的表达情况.结果 原代骨髓间充质干细胞呈集落状生长,细胞呈梭形、纺锤形,呈放射状生长,传代后呈均一的成纤维细胞样.骨髓间充质干细胞生长性状相对稳定,1、3、5代细胞生长曲线基本一致.流式细胞仪鉴定表明,骨髓间充质干细胞CD44、CD29表达呈阳性,CD45、CD38表达呈阴性.结论 采用密度梯度离心法结合贴壁培养法能获得纯度较高的骨髓间充质干细胞,并且在体外培养条件下可大量增生,形成形态均一的细胞集落,可以作为组织工程中种子细胞的来源.     

骨髓基质细胞体内成骨分化研究

目的探讨将自体骨髓基质干细胞（BMSCs）移植到股骨头缺损坏死部位后能否成活、增殖,并向成骨分化,促进骨修复。方法取狗犬髂骨骨髓,体外培养扩增BMSCs,经5-溴脱氧尿嘧啶核苷（BrdU）标记,移植到股骨头骨缺损模型。术后5周取材。行大体组织观察,组织化学染色,免疫组化检测骨钙素、骨桥素。结果BMSCs能增加骨缺损区内的成骨量、提高骨基质矿化的程度和骨成熟度,提高成骨细胞分化特异性标记物骨钙素、骨桥素的表达。新骨形成越活跃的地方,BrdU阳性细胞数目越多,很多成骨细胞BrdU阳性,新骨形成区的很多血管壁也分布着BrdU阳性细胞。结论移植的骨髓细胞在股骨头内能成活,并能停留在骨坏死、缺损部位生长增殖。移植的BMSCs能分化成为多种细胞,包括成骨细胞、血管壁细胞等。BMSCs不但能直接通过分化成成骨细胞参与成骨,也能通过参与血管形成而促进成骨。     

Donor hematopoietic progenitor cells in nonmyeloablated rat recipients of allogeneic bone marrow and liver grafts

BACKGROUND: Although the persistence of multilineage microchimerism in recipients of long-surviving organ transplants implies engraftment of migratory pluripotent donor stem cells, the ultimate localization in the recipient of these cells has not been determined in any species. METHODS: Progenitor cells were demonstrated in the bone marrow and nonparenchymal liver cells of naive rats and in Brown Norway (BN) recipients of Lewis (LEW) allografts by semiquantitative colony-forming unit in culture (CFU-C) assays. The LEW allografts of bone marrow cells (BMC) (2.5x10(8)), orthotopic livers, or heterotopic hearts (abdominal site) were transplanted under a 2-week course of daily tacrolimus, with additional single doses on days 20 and 27. Donor CFU-C colonies were distinguished from recipient colonies in the allografts and recipient bone marrow with a donor-specific MHC class II monoclonal antibody. The proportions of donor and recipient colonies were estimated from a standard curve created by LEW and BN bone marrow mixtures of known concentrations. RESULTS: After the BMC infusions, 5-10% of the CFU-C in the bone marrow of BN recipients were of the LEW phenotype at 14, 30, and 60 days after transplantation. At 100 days, however, donor CFU-C could no longer be found at this site. The pattern of LEW CFU-C in the bone marrow of BN liver recipients up to 60 days was similar to that in recipients of 2.5x10(8) BMC, although the donor colonies were only 1/20 to 1/200 as numerous. This was expected, because the progenitor cells in the passenger leukocytes of a single liver are equivalent to those in 1-5x10(6) BMC. Using a liquid CFU-C assay, donor progenitor cells were demonstrated among the nonparenchymal cells of liver allografts up to 100 days. In contrast, after heart transplantation, donor CFU-C could not be identified in the recipient bone marrow, even at 14 days. CONCLUSION: effective immunosuppression, allogeneic hematopoietic progenitors compete effectively with host cells for initial engraftment in the bone marrow of noncytoablated recipients, but disappear from this location between 60 and 100 days after transplantation, coincident with the shift of donor leukocyte chimerism from the lymphoid to the nonlymphoid compartment that we previously have observed in this model. It is possible that the syngeneic parenchymal environment of the liver allografts constitutes a privileged site for persistent progenitor donor cells.     

Interaction of allogeneic demineralized bone matrix and porous hydroxyapatite bioceramics in lumbar interbody fusion in rabbits

Bone repair by autograft is effective in clinical practice. However, serious problems arise when a considerable volume of transplant is needed, as with spinal fusion procedures. The use of bone substitutes combined with osteoinductive agents may contribute to the solution of such problems. In this study, the effectiveness of such a procedure was tested in an experimental model of interbody fusion in rabbits in which the incorporation of a porous hydroxyapatite block (HA) was enhanced by the addition of allogeneic demineralized bone matrix (DBM). The latter was used as a delivery system for the osteoinductive activity of the bone morphogenetic protein contained in the matrix. A group implanted with combined HA + DBM showed significantly earlier stabilization of the fusion when compared to groups implanted with DBM alone, HA alone, and bone autografts. On the other hand, the general results of the fusion with HA + DBM were superimposable on those of autografts. With further research, the combination of a bone substitute and an osteoinductive agent may constitute an alternative to the use of bone autografts.     

Osteogenic differentiation of human bone marrow-derived mesenchymal cells cultured on alumina ceramics

Alumina ceramics have excellent mechanical and biocompatible properties, but are bioinert and hence have no bone-bonding properties. We took a tissue-engineering approach in an attempt to modify the ceramic surface and so provide an osteogenic/osteoconductive milieu. We obtained human bone marrow mesenchymal cells from four donors and then cultured the cells for two weeks on alumina ceramic in the presence of beta-glycerophosphate, ascorbic acid and dexamethasone. The cells showed extensive alkaline phosphatase staining and mineralization, as evidenced by Alizarin Red S staining and calcein uptake. Biochemical analyses revealed high levels of alkaline phosphatase activity, osteocalcin expression and calcium content. This data indicates the appearance of active osteoblasts that are concomitant with bone matrix formation, i.e., in vitro cultured bone. The cultured bone/alumina composites should prevent the aseptic loosening of all-alumina ceramic joints or the detachment of implanted alumina ceramics, and thus could have clinical significance in orthopedic reconstructive surgery.     

Osteogenic stem cells and the stromal system of bone and marrow

According to current hypothesis, cells of the osteogenic lineage, which includes both osteoblasts and chondroblasts, are derived from a stromal stem cell in the postnatal organism. That there exist osteogenic precursors in association with the soft, fibrous tissue of the marrow stroma is well established. An osteogenic tissue comprised of cartilage and bone is formed when marrow or marrow cell suspensions are cultured in vivo within diffusion chambers. Bone with a functional marrow organ is formed when marrow or marrow cell suspensions are transplanted heterotopically, e.g., under the renal capsule. Cultures of marrow stromal fibroblasts are readily established in vitro from single-cell bone marrow suspensions. Such cultures do not demonstrate overt differentiation in an osteogenic direction in vitro. When transplanted in vivo, however, they differentiate to form cartilage and bone in diffusion chambers and bone with a functional marrow organ when transplanted heterotopically. Single-cell bone marrow suspensions can be cultured in vitro under conditions that facilitate the formation of stromal fibroblast colonies. Circumstantial evidence supports the conclusion that each colony is derived from a single initiating cell termed a colony-forming unit-fibroblastic (CFU-F). A proportion of CFU-F demonstrates extensive proliferative potential both in vitro and in vivo. In vitro the extensive proliferative potential of a subset of CFU-F has been shown to be associated with a capacity for extensive self-renewal. On transplantation in vivo, the progeny of a proportion of CFU-F has been shown to be capable of proliferating and differentiating into all the stromal cell lines necessary for the formation of bone and reconstitution of the hematopoietic inductive microenvironment. These findings provide strong circumstantial evidence to support the hypothesis that there are stem cells present within the marrow stroma that are capable of giving rise to cells of a number of different lineages, including those of the osteogenic lineage (chondroblasts and osteoblasts).     

Culture-expanded periosteal-derived cells exhibit osteochondrogenic potential in porous calcium phosphate ceramics in vivo.

Chick tibial periosteal cells were enzymatically disaggregated, introduced into cell culture, and subcultured. These subcultured cells were combined with porous calcium phosphate ceramics and implanted into a subcutaneous site in athymic mice as an immunocompatible host to test the in vivo osteochondrogenic potential of this composite graft. These cells eventually gave rise to bone tissue in the pores of ceramics at the heterotopic implantation sites. The process of bone formation occurred through two different mechanisms: Intramembranous bone formation occurred at the peripheral pores of ceramics early, and endochondral bone formation occurred in the central pores later. Cultured chick muscle fibroblasts of the same-aged donor as controls did not form bone or cartilage under identical conditions to those of cultured periosteal-derived cells. These results raise the possibility that composite graft of cultured periosteal-derived cells and porous ceramics can be clinically used as a bone graft substitute in situations requiring bone augmentation or regeneration.     

淋巴因子激活的骨髓细胞对小鼠同种异体骨髓移植的影响

以致死性剂量照射的成年小鼠为受鼠进行同种异体骨髓移植（Ａｌｌｏ－ＢＭＴ），实验组小鼠观察期间平均存活天数较对照组明显延长（Ｐ＜０．００１），应用肿瘤移植及单向混合淋巴细胞培养（ＭＬＣ）二项指标证实Ａｌｌｏ－ＢＭＴ的部分植活。实验结果表明，淋巴因子激活的骨髓细胞（ＬＡＢＭＣ）可有效预防致死性移植物抗宿主病（ＧＶＨＤ），且不影响供髓植活。     

Engraftment of allogeneic dog bone marrow.

Resistance to allogeneic bone-marrow grafts (AR) was found to occur in many species, including the dog. The i.v. administration of silica particles suppressed AR in vivo in this species. Genetic studies provide suggestive evidence for the existence of a previously unrecognized system or systems in the canine major histocompatibility complex controlling AR.