Biocompatibility testing of different sterilised or disinfected allogenous bone grafts in comparison to the gold standard of autologous bone grafts--an "in vitro" analysis of immunomodulation

INTRODUCTION: Repair of large skeletal defects using bone allografts has become a routine procedure in orthopaedic and trauma surgery. Different procedures of sterilisation (82.5 degrees C disinfection; 121 degrees C autoclaving; PES; Tutoplast; 25 kGy gamma irradiation) are available to inactivate bacteria and fungi, including their spores, as well as viruses in human bone allografts. The efficiency of these procedures has been proven. However, the effects on the cellular response are rarely investigated. This present in vitro study investigates the immunological answer of human bone marrow cells to human allogenous and autologous bone platelets which were sterilised by different methods. MATERIALS AND METHODS: Human bone marrow cells and the bone platelets were harvested from patients undergoing a total hip replacement. All patients provided informed consent. Human bone platelets, 10 mm in diameter, 3 mm in height, were produced from femoral heads which were removed within the scope of total hip replacements. They were sterilised by different procedures or were disinfected (gamma radiotherapy, PES/ethanol treatment, Tutoplast procedure, 121 degrees C autoclaving, > 82.5 degrees C thermodisinfection). In addition, an autologous in vitro bone donation was simulated and compared with the allogenous bone grafts. Endobon was evaluated as a bovine hydroxyapatite ceramic. As control a human bone marrow cell culture without bone platelets was used. Over a period of four weeks the changes of the immunogenic cell populations were analysed in vitro (FACS analysis). Light and scanning microscopy were done to reveal morphological differences. As a vitality test the trypan-blue staining was performed. RESULTS: Light and scanning microscopy demonstrated large differences between the various sterilisation and disinfection methods. After 4 weeks the autologous bone platelets were completely covered with homogenously distributed human osteoblast like cells. The heat-sterilised/disinfected transplants demonstrated similar effects compared to the autologous bone grafts while the irradiated bone platelets demonstrated less cell coverage. 2/3 of the cells were vital on average after four weeks, with the exception of the irradiated bone platelets. The FACS analysis revealed in comparison to the control group provable differences in the immunological answer for the autologous bone donation as well as for the differently sterilised or disinfected allogenous bone grafts. The heat sterilisation or, respectively, disinfection methods compared to the autologous bone donation demonstrated almost similar in vitro effects. By far the worst results, characterised by an excessively increased portion of cytotoxic T-cells and a decreased amount of viable cells, were seen in the 25 kGy gamma irradiation samples. CONCLUSIONS: The results demonstrate the influence of the different sterilisation and disinfection procedures on the differentiation of human marrow cells (host). Similar in vitro effects were seen for the autologous and heat-treated bone platelets. The treatment of allogenous bone grafts with PES/ethanol and the Tutoplast procedures showed, just as Endobon, only low differences in comparison with the control cultures. The worse results in the case of the irradiated bone platelets may be explained by the production of free radicals which led to an excessive cell death.     

Einfluß verschiedener Knochendesinfektions- und Sterilisationsverfahren auf die Osteoblastenfunktion Eine vergleichende In-vitro-Studie

All sterilization and disinfection procedures for bone grafts are different in regard to influence of bone graft features, which may influence the function of different cell types. We used an in-vitro approach to assess the influence of bone matrix, which was sterilized or disinfected, on osteoblastic activities in-vitro by simulating a cell-transplant-interface. Primary bovine osteoblast cell cultures were established from periosteum. Bone graft specimens made of bovine cortical bone (O 15 mm, 300 microns thickness) were treated in 5 different ways: autoclaved, ethylene-oxide-sterilized, demineralized and low-temperature-plasma-sterilized (DEM-LTP), chemically sterilized (modified Tutoplast method), and 80 degrees C-temperature disinfected. The following cell function parameters were assayed: plating efficiency proliferation by measuring the DNA-content, and MTT-activity, soluble protein and extracellular matrix synthesis, alkaline phosphatase, and osteocalcin expression. All disinfected bone grafts were biocompatible with primary periosteal osteoblasts. Measured cell activities upon bone specimens showed better results than cells of the plastic surface control. The DEM-LTP-bone showed better results in comparison to other groups, and stimulated the proliferation and differentiation.     

Hyaluronan-based biomaterial (Hyaff-11) as scaffold to support mineralization of bone marrow stromal cells

Various techniques are widely used to repair bone defects, association of hyaluronan-based biodegradable polymers (Hyaff-11) with bone marrow stromal cells (BMSC) promises to provide successful cell scaffolds for tissue-engineered repair of bone tissue. We evaluate in vitro and in vivo the potential of Hyaff-11 to facilitate mineralization of BMSC. Rat BMSC were seeded on Hyaff-11 and their differentiation were assessed at different time points. Osteogenic differentiation was investigated in vitro analysing the expression of alkaline phosphatase and osteocalcin. Mineralization of bone defects was evaluated also in vivo implanting Hyaff-11 scaffold combined with BMSC in large segmental radius defects. In vitro, we found a decrease expression of alkaline phosphatase and an increase of osteocalcin. In vivo, our data showed that mineralization was induced and basic fibroblast growth factor contributed to this process. These results provide a demonstration to therapeutic potential of Hyaff-11 as appropriate carrier vehicle for differentiation and mineralization of BMSC and for the repair of bone defects.     

Cellular compatibility of improved scaffold material with deproteinized heterogeneous bone

Objective： To study cellular compatibility of improved scaffold material with deproteinized heterogeneous bone and provide experimental basis on choosing the scaffold material in bone tissue engineering. Methods： Bone marrow stromal cells （BMSC） were co-cultured with heterogeneous deproteinized bone in vitro. The contrast phase microscope, scanning electron microscope, MTT assay, flow cytometry were performed and the BGP content and ALP activities were detected in order to observe the cell growth, adhesion in the material, cell cycle and cell viability. Results. The scaffold material of deproteinized heterogeneous bone had no inhibitory effect on cellular proliferation, differentiation and secretion function of BMSCs. Conclusions ： The established heterogeneous deproteinized bone has good biocompatibility with BMSCs and is a potentially ideal scaffold material for bone tissue engineering.     

Induction of bone tissue on different matrices: an in vitro and a in vivo pilot study in the SCID mouse

AIM: Three resorbable biomaterials were evaluated regarding proliferation and osteogenic differentiation of human bone marrow stromal cells (BMSC) in vitro. In a second step, the new biomaterial, calcium-deficient hydroxyapatite (CDHA), was tested in a pilot in vivo study by subcutaneous implantation in the severe combined immunodeficiency (SCID) mouse. METHODS: CDHA, beta-tricalcium phosphate (beta-TCP), and demineralized bone matrix (DBM) were seeded with human BMSC and cultured in osteogenic supplements for 3 weeks. In the pilot in vivo study, CDHA was seeded with BMSC and kept in osteogenic media for 2 weeks (group A) before subcutaneous implantation in 8 SCID mice for 3 and 8 weeks. In addition, CDHA seeded with BMSC without prior osteogenic induction (group B) and empty ceramics were implanted in each mouse. RESULTS: Total protein content and the values for specific alkaline phosphatase (ALP) increased significantly in vitro on all matrices, but no significant difference between the groups was noted. In the pilot in vivo study all ceramics were well penetrated by cells. After 8 weeks 2 of 4 samples in group B and 1 of 4 samples in group A revealed cells resembling hypertrophic chondrocytes. Specific ALP was higher in the group B (p = 0.012, Z = - 2.5) compared to empty ceramics. There were no significant differences between groups A and B. Differences between group A and the empty control did not become significant (p = 0.069, Z = - 1.8). CONCLUSION: All three matrices promoted BMSC proliferation and differentiation to osteogenic cells in vitro. Human BMSC on CDHA showed signs of osteogenic differentiation after subcutaneous implantation into SCID mice.     

MC3T3‐E1 Cells Behavior on Surfaces Bombarded by Argon Ions in Planar Cathode Discharge

To evaluate the effect of topography in nanoscale, titanium surfaces were bombarded by argon ions (a chemically inert gas), in an atmosphere of plasma. The effects of surface parameters on morphology, adhesion, proliferation, and MC3T3‐E1 preosteoblasts differentiation were analyzed. Nontreated (smooth) surfaces were used as a control. The levels of average roughness (Ra) observed in bombarded and smooth titanium surfaces were of 95 and 14 nm, respectively. The wettability increased on treated surfaces. The number of attached cells (30 and 60 min) was significantly higher on the bombarded surface. The cell proliferation after 3 and 7 days was also significantly higher on the ion‐bombarded surface. In addition, the ALP activity and expression of osteocalcin were higher in cells grown on the treated surface. The results showed that bombardment with argon ions increased the roughness and the wettability of the Ti surface, promoting a significant increase in the adhesion, proliferation, and differentiation of preosteoblasts.     

Glucocorticoids impair bone formation of bone marrow stromal stem cells by reciprocally regulating microRNA-34a-5p

Summary

The inhibitory effects of glucocorticoids (GCs) on bone marrow stromal stem cell (BMSC) proliferation and osteoblastic differentiation are an important pathway through which GCs decrease bone formation. We found that microRNA-34a-5p was a critical player in dexamethasone (Dex)-inhibited BMSC proliferation and osteogenic differentiation. MicroRNA-34a-5p might be used as a therapeutic target for GC-impaired bone formation.

Introduction

The inhibitory effects of glucocorticoids (GCs) on bone marrow stromal stem cell (BMSC) proliferation and osteoblastic differentiation are an important pathway through which GCs decrease bone formation. The mechanisms of this process are still not completely understood. Recent studies implicated an important role of microRNAs in GC-mediated responses in various cellular processes, including cell proliferation and differentiation. Therefore, we hypothesized that these regulatory molecules might be implicated in the process of GC-decreased BMSC proliferation and osteoblastic differentiation.

Methods

Western blot, quantitative real-time PCR, and cell proliferation and osteoblastic differentiation assays were employed to investigate the role of microRNAs in GC-inhibited BMSC proliferation and osteoblastic differentiation.

Results

We found that microRNA-34a-5p was reciprocally regulated by Dex during the process of BMSC proliferation and osteoblastic differentiation. Furthermore, we confirmed that microRNA-34a-5p was a critical player in Dex-inhibited BMSC proliferation and osteogenic differentiation. Mechanistic studies showed that Dex inhibited BMSC proliferation by microRNA-34a-5p targeting cell cycle factors, including CDK4, CDK6, and Cyclin D1. Furthermore, downregulation of microRNA-34a-5p by Dex leads to Notch signaling activation, resulting in inhibition of BMSC osteogenic differentiation.

Conclusions

These results showed that microRNA-34a-5p, a crucial regulator for BMSC proliferation and osteogenic differentiation, might be used as a therapeutic target for GC-impaired bone formation.

     

大鼠损伤脊髓提取液对骨髓基质细胞培养特性的影响

目的：探讨损伤脊髓提取液对骨髓基质细胞增殖和分化的影响。方法：将正常、伤后1周及伤后6周脊髓提取液加入细胞培养基中，以四唑盐法及免疫组化方法观察其对骨髓基质细胞增殖及抗原表达的影响。结果：加入脊髓提取液后对骨髓基质细胞的增殖无明显影响，但抗原表达发生变化，纤维连接蛋白(FN)表达减弱，且出现神经丝蛋白(NF)和胶质纤维酸性蛋白(GFAP)的表达，细胞形态出现神经细胞样改变。结论：骨髓基质细胞可能是一种脊髓损伤修复的理想细胞。     

骨髓间充质干细胞外泌体在骨质疏松中的研究进展

骨质疏松症(osteoporosis,OP)的发病与骨代谢平衡的破坏有关,骨吸收作用增强,骨形成作用不足,骨稳态发生改变,均会引起骨质疏松。成骨不足与骨髓间充质干细胞(bone marrow-derived mesenchymal stem cell,BMSC)分化方向密切相关。BMSC向成骨分化方向减少、成脂分化增多是骨质疏松症发病的重要机制,因此,寻找决定BMSC分化方向的关键因子为OP的研究和治疗提供了新思路。外泌体是由细胞分泌到胞外的膜性囊泡,富含不同种类的核酸、蛋白质、脂质和信号分子等多种生物学活性物质,参与调节和完成细胞间的信息交流与传递,影响胞内信号转导发挥生物学功能。研究表明, BMSC来源的外泌体可有效改善骨质疏松症状,促进BMSC增殖、成骨分化及骨再生。本文对BMSC来源的外泌体应用于OP的研究进展进行综述,旨在为今后的研究提供参考和思路。     

静水压力刺激对人骨髓间充质干细胞成骨、成脂双向分化的影响

目的 研究短期和较长期的静水压力负荷对人骨髓间充质干细胞(BMSC)成骨或成脂的影响,并探讨细胞外信号调节激酶(ERK)1/2信号通路在其中作用.方法 对BMSC施加40或80 kPa的静水压强,分别持续1 h或4 h,然后加入10 mmol/L U0126阻断ERK1/2信号通路,通过半定量RTPCR和实时定量PCR方法检测成骨标志基因Cbfa1和骨钙素(OC)以及成脂标志性基因PPARγ2和Adipsin的mRNA表达水平.对BMSC施加40 kPa的静水压刺激,每天作用4 h,7 d和14 d后检测Cbfa1、OC、PPARγ2和Adipsin的mRNA表达水平,在3、7、14 d通过组织化学染色和定量测定方法检测碱性磷酸酶的活性和表达量.结果 40 kPa压强的静水压连续刺激4 h可以促进Cbfa1、OC的mRNA表达和抑制PPARγ2、Adipsin的mRNA表达.40 kPa的静水压连续作用7 d,仍表现为对BMSC的促成骨分化作用,然而连续作用14 d后,则表现为促成脂分化作用.成骨诱导条件并不协同静水压的促BMSC成骨分化作用.而U0126并不能完全抑制BMSC对静水压刺激的反应.结论 一定强度和作用时间的静水压刺激可以促进BMSC的成骨分化,但较长期的作用可能会促进成脂分化.成骨诱导培养条件与静水压的促BMSC成骨分化作用无协同作用.     

Deep-freezing versus 4 degrees preservation of avascular osteocartilaginous shell allografts in rats

Osteocartilaginous allografts (distal femurs of rats) were stored at 4 degrees for six, 12, 24, and 48 hours and at -80 degrees for five days and then evaluated for viability of the bone and cartilage. Storage at 4 degrees for 12 or 24 hours had little effect on cartilage viability but decreased bone viability to 40% and 10% of controls, respectively. Storage at -80 degrees for five days resulted in nonviable bone in all cases but showed an either/or response of cartilage, with high viability in two cases and nonviability in the other eight cases. In a second set of experiments, femurs from rats were stored in situ at 4 degrees for 12 or 24 hours or were harvested and stored at -80 degrees for five days, after which they were transplanted into rats of a different strain. The antibody response to each set of femurs was measured at two, six, and 12 weeks after operation. The 4 degrees storage resulted in a moderately decreased immunogenicity, whereas the storage at -80 degrees resulted in significantly reduced immunogenicity.     

Three isolation techniques for primary culture of human osteoblast-like cells: a comparison

The culture of osteoblast-like cells of human origin has become an important experimental model in bone biology. We report here a comparison and evaluation of three of the most widely used systems available today: bone marrow stroma cell cultures (BMSC), human osteoblast explant cultures (hOB) and osteoblast explant cultures from collagenase-treated bone (hOBcol). Cultures from 16 bone specimens obtained from various donors were established and their expression of the osteoblast phenotype were then compared in secondary cultures by use of biochemical markers. BMSC had the highest basal and 1,25-dihydroxyvitaminD3 (1,25(OH)2D3)-induced alkaline phosphatase activities in all cell isolations, with levels approximately twice those in explant cultures. Basal osteocalcin secretion was low-to-undetectable in all cell cultures but was detected in 1,25(OH)2D3-stimulated cultures. BMSC produced half of the amount of osteocalcin synthesized in explant cultures. The BMSC cultures also synthesized the lowest amounts of type I collagen, whereas collagen type III synthesis did not differ significantly among the various cultures. When secondary cultures were treated with 100 nM dexamethasone in the presence of ascorbic acid (50 microg/mL) and beta-glycerophosphate (10 mM), cultures deposited calcium mineral into the cell layer within 2-4 weeks. PTH-induced cAMP formation was detected in only 5 of 15 isolations and no consistent isolation-dependent response pattern was seen. We conclude that BMSC cultures differ significantly from explant cultures obtained from the same bone specimen. However, all cultures represent cells which can differentiate further and induce mineralization of the extracellular matrix in response to osteoinductive drugs.     

Three isolation techniques for primary culture of human osteoblast-like cells: A comparison

The culture of osteoblast-like cells of human origin has become an important experimental model in bone biology. We report here a comparison and evaluation of three of the most widely used systems available today: bone marrow stroma cell cultures (BMSC), human osteoblast explant cultures (hOB) and osteoblast explant cultures from collagenase-treated bone (hOB^col). Cultures from 16 bone specimens obtained from various donors were established and their expression of the osteoblast phenotype were then compared in secondary cultures by use of biochemical markers. BMSC had the highest basal and 1,25-dihy-droxyvitaminD₃ (1,25(OH)₂D.))-induced alkaline phosphatase activities in all cell isolations, with levels approximately twice those in explant cultures. Basal osteocalcin secretion was low-to-undetectable in all cell cultures but was detected in 1,25(OH)₂D₃-stimulated cultures. BMSC produced half of the amount of osteocalcin synthesized in explant cultures. The BMSC cultures also synthesized the lowest amounts of type I collagen, whereas collagen type III synthesis did not differ significantly among the various cultures. When secondary cultures were treated with 100 nM dexam-ethasone in the presence of ascorbic acid (50 u.g/mL) and B-glycerophosphate (10 mM), cultures deposited calcium mineral into the cell layer within 2-4 weeks. PTH-induced cAMP formation was detected in only 5 of 15 isolations and no consistent isolation-dependent response pattern was seen. We conclude that BMSC cultures differ significantly from explant cultures obtained from the same bone specimen. However, all cultures represent cells which can differentiate further and induce mineralization of the extracellular matrix in response to osteoinductive drugs.     

Wirkung verschiedener Verfahren zur Keiminaktivierung auf die BMP-Aktivität von Knochengewebe

The sterilisation of allogeneic bank bone without significant loss of biological transplant activity makes high demands on the procedure used. For reliable sterilisation of hard tissues it is necessary to use harsh chemical and/or physical methods, which on the other hand should not exert significant adverse effects on the biological properties of the transplants. The effects of disinfection by moderate heat treatment, sterilization by γ-irradiation and low temperature conservation on osteoinductive factors in bone matrix were examined comparatively in an experimental animal study. The quantitative determination of residual BMP activity demonstrated a significant loss of 50% of starting activity by heat disinfection at 80°C. Irradiated matrix implants revealed a non significant reduction of only 6% of initial activity and frozen bone showed a slightly increased activity of 10%. Among the procedures currently used γ-irradiation seems to be most appropriate because of its known strong sterilization effects and preservation of the biological activity of bone transplants based on a pronounced resistance of BMP to irradiation.     

兔骨髓基质干细胞体外分离培养及生物学特性研究

目的：研究兔骨髓基质干细胞（bone marrow-derived mesenchymal stem cells,BMSC）体外分离、诱导培养及增殖特点,探讨其生物学特性。方法：抽取兔骨髓,密度梯度离心结合贴壁培养法分离BMSC,诱导培养液定向诱导传代细胞向成骨细胞分化。倒置显微镜下观察细胞生长及增殖情况,流式细胞仪检测细胞表面标志物的表达,免疫组织化学观察Ⅰ型胶原表达,并检测碱性磷酸酶活性及细胞矿化作用。结果：体外培养的兔BMSC贴壁生长,10～14天后形成克隆。细胞形态为均一的长梭形并呈漩涡状排列,流式细胞仪检测CD34,CD45阴性,CD44阳性。免疫组化可检测到Ⅰ型胶原表达,碱性磷酸酶活性明显增高,并且出现矿化结节。结论：密度梯度离心结合贴壁培养BMSC,操作简单,细胞易于成活,诱导条件下成骨能力肯定,适合作为骨组织工程的种子细胞。     

In vivo study on the healing of bone defects treated with bone marrow stromal cells, platelet-rich plasma, and freeze-dried bone allografts, alone and in combination.

The repair of confined trabecular bone defects in rabbits treated by autologous bone marrow stromal cells (BMSC), platelet-rich plasma (PRP), freeze-dried bone allografts (FDBA) alone and in combination (BMSC + PRP; FDBA + BMSC; FDBA + PRP; FDBA + PRP + BMSC) was compared. A critical size defect was created in the distal part of the femurs of 48 adult rabbits. Histology and histomorphometry were used in the evaluation of healing at 2, 4, and 12 weeks after surgery. The healing rate (%) was calculated by measuring the residual bone defect area. Architecture of the newly formed bone was compared with that of bone at the same distal femur area of healthy rabbits. The defect healing rate was higher in PRP + BMSC, FDBA + PRP, FDBA + BMSC, and FDBA + PRP + BMSC treatments, while lower values were achieved with PRP treatment at all experimental times. The highest bone-healing rate at 2 weeks was achieved with FDBA + PRP + BMSC treatment, which resulted significantly different from PRP (p < 0.05) and BMSC (p < 0.05) treatments. At 4 weeks, the bone-healing rate increased except for PRP treatment. Finally, the bone-healing rate of FDBA + PRP, FDBA + BMSC, and FDBA + PRP + BMSC was significantly higher than that of PRP at 12 weeks (p < 0.05). At 12 weeks, significant differences still existed between PRP, BMSC, and FDBA groups and normal bone (p < 0.05). These results showed that the combination of FDBA, BMSC and PRP permitted an acceleration in bone healing and bone remodeling processes.     

pcDNA_(3.1)-OGP基因转染兔骨髓基质干细胞的表达及生物学效应的观察

[目的]观察成骨生长肽(osteogenic growth peptid,OGP)基因转染兔骨髓基质干细胞后的表达及表达产物对骨髓基质干细胞向成骨细胞分化的影响.[方法]构建重组真核表达载体pcDNA_(3.1)-OGP,并在脂质体介导下,将其导入兔骨髓基质干细胞.通过G418筛选获得阳性克隆.用RT-PCR方法榆测OGP基因在骨髓基质干细胞内的表达.Ⅰ型胶原和碱性磷酸酶的检测观察转染pcDNA_(3.1)-OGP后骨髓基质干细胞向成骨细胞分化情况.[结果]成功构建真核表达载体pcDNA_(3.1)-OGP,用RT-PCR方法及碱性磷酸酶和Ⅰ型胶原检测证实OGP基因能在骨髓基质干细胞中表达并促进其向成骨细胞分化.[结论]经pcDNA_(3.1)-OGP转染的兔骨髓基质干细胞不仅可以表达OGP,而且具有向成骨细胞系分化的特性.     

Alveolar bone marrow as a cell source for regenerative medicine: differences between alveolar and iliac bone marrow stromal cells.

We isolated and expanded BMSCs from human alveolar/jaw bone at a high success rate (70%). These cells had potent osteogenic potential in vitro and in vivo, although their chondrogenic and adipogenic potential was less than that of iliac cells. INTRODUCTION: Human bone marrow stromal cells (BMSCs) have osteogenic, chondrogenic, and adipogenic potential, but marrow aspiration from iliac crest is an invasive procedure. Alveolar BMSCs may be more useful for regenerative medicine, because the marrow can be aspirated from alveolar bone with minimal pain. MATERIALS AND METHODS: In this study, alveolar bone marrow samples were obtained from 41 patients, 6-66 years of age, during the course of oral surgery. BMSCs were seeded and maintained in culture with 10% FBS and basic fibroblast growth factor. In addition, BMSCs were induced to differentiate into osteoblasts, chondrocytes, or adipocytes in appropriate medium. RESULTS AND CONCLUSION: From a small volume (0.1-3 ml) of aspirates, alveolar BMSCs expanded at a success ratio of 29/41 (70%). The success rate decreased with increasing donor age, perhaps because of age-dependent decreases in the number and proliferative capacity of BMSCs. The expanded BMSCs differentiated into osteoblasts under osteogenic conditions in 21-28 days: the mRNA levels of osteocalcin, osteopontin, and bone sialoprotein, along with the calcium level, in alveolar BMSC cultures were similar to those in iliac cultures. However, unlike iliac BMSC, alveolar BMSC showed poor chondrogenic or adipogenic potential, and similar differences were observed between canine alveolar and iliac BMSCs. Subsequently, human alveolar BMSCs attached to beta-tricalcium phosphate were transplanted into immunodeficient mice. In transplants, new bone formed with osteoblasts and osteocytes that expressed human vimentin, human osteocalcin, and human GAPDH. These findings suggest that BMSCs have distinctive features depending on their in vivo location and that alveolar BMSCs will be useful in cell therapy for bone diseases.