Influence of harvesting technique and donor site location on in vitro growth of osteoblastlike cells from facial bone

PURPOSE: Donor morbidity is minimized when tissue engineering is applied to produce osteogenic grafts by growing osteoblasts on biomaterials. However, limiting factors are the origin, proliferation, and differentiation of osteoblasts. Therefore, the aim of this study was to evaluate the efficacy of growing osteoblasts from different types of bone samples and to assess the influence of the donor site. MATERIALS AND METHODS: From 28 patients 37 bone specimens were obtained during removal of third molars in the maxilla and mandible. Seventeen specimens were bone chips and 20 were bone sludge. After subculturing primary cultures, histochemical and immunhistochemical tests (EZ4U test, BrdU labeling, ALP histochemistry, type I collagen immunohistochemistry, osteocalcin ELISA) were performed to determine cell proliferation, viability, and differentiation. RESULTS: Both bone chips and bone sludge from the mandible and maxilla are suitable for culturing human osteoblastlike cells. However, bone chips were superior to bone sludge with respect to ability to grow cells, and maxillary bone was superior to mandibular bone in this regard. Harvesting technique had only little influence on the expression of cell differentiation markers (ALP, type I collagen, osteocalcin). DISCUSSION AND CONCLUSION: Chips from human membrane bone, especially from the maxilla, are suitable for culturing high numbers of differentiated osteoblastlike cells. These cells may be used to tissue engineer bone grafts, which may be used to enhance the implant placement site.     

Effects of cortical bone perforation on experimental guided bone regeneration

This study was designed to evaluate the effects of cortical bone perforation histologically and histomorphometrically on guided bone regeneration (GBR) in rabbits. After elimination of the periosteum, cortical bone defects of two sizes were made in the external cortical plate of the frontal bone (Group A: 1 x 15 mm; Group B: 3 x 15 mm). A non-resorbable membrane filled with autogenous blood was placed in the experimental area and secured with titanium pins. After 1 and 2 weeks, vascularized connective tissue and new bone were generated in the space surrounding the defects in both the groups. The amount of vascularized connective tissue generated in Group B was greater than that in Group A at 1 week. Alkaline phosphatase (ALP) was expressed on the bone surrounding the perforation. The expression of ALP was more extensive in Group B than in Group A and was proportional to the breadth of perforation. At 2 weeks, the perforated region was almost covered with new bone in Group A. ALP was expressed at the periphery of newly formed bone. The expression of ALP was proportional to the breadth and height of perforation. At 6 weeks, semicircular outgrowth of bone towards the periphery of the perforated region was observed in both the groups. Newly formed bone volume and ALP expression in Group B were more extensive than those in Group A. At 12 weeks, the space was filled with bone and connective tissue in both the groups. There was no difference in ALP expression between Groups A and B. Histomorphometric analysis showed significant differences between both the groups (two-way ANOVA, P<0.01). We conclude that a larger perforation is associated with prompter bone formation in the secluded space during GBR.     

富血小板血浆PRP的体外骨诱导作用研究

目的 :探讨在体外培养中富血小板血浆 (Platelet -RichPlasma ,PRP)对骨髓基质细胞诱导成骨的影响。方法 :从自体静脉血中提取PRP ,配制成条件培养液 ,并作用于培养状态的骨髓基质细胞 ,染色检测细胞碱性磷酸酶表达情况 ,钙化结节形成率 ,放免法测定培养液中骨钙素的含量。结果 :骨髓基质细胞经诱导培养后 ,碱性磷酸酶、骨钙素表达明显增加。结论 :体外培养时 ,PRP可促进骨髓基质细胞的成骨分化。     

Clinical evaluation of particulate allogeneic with and without autogenous bone grafts and resorbable collagen membranes for bone augmentation of atrophic alveolar ridges

Objectives: The evaluate the clinical outcome of bone augmentation with the use of particulate mineralized freeze‐dried bone allograft (FDBA) with or without the addition of autogeneous bone chips, applied in a bi‐layered (BL) technique, covered by a resorbable cross‐linked collagen membrane. Material and methods: Fifty patients presenting with a vertical and/or lateral ridge deficiency of at least 3 mm were included: Group FDBA, N=27 patients, particulate FDBA was the only graft; and Group BL, N=23 patients, a BL bone grafting procedure where autogenous bone chips were the inner layer and FDBA the outer. Bone graft was covered with a ribose cross‐linked collagen barrier membrane. Ridge dimensions were clinically or radiographically (computerized tomography scan) measured at the time of the bone augmentation procedure and at implant placement or uncovering and the maximum linear vertical or horizontal calcified tissue gain was calculated. Statistical analysis consisted of linear regression analysis, with maximum bone gain being the dependent variable. Results: In the FDBA group, mean vertical bone gain was 3.47 mm (SD 1.25) and the horizontal, 5 mm (SD 1.28), while in the BL values were 3.5 mm (SD 1.2) and 3.6 mm (SD 1.72), respectively. Addition of autogenous bone does not appear to statistically significantly enhance the results. Spontaneous membrane exposure occurred in 24% of the cases and was the only variant that significantly influenced results (P<0.001). Conclusions: Large vertical and/or horizontal ridge deficiencies may be treated with FDBA and ribose cross‐linked collagen barrier membranes with good clinical outcome. No added effect of the application of a layer of autogenous bone in these bone augmentation procedures could be demonstrated. Spontaneous membrane exposure was the only parameter to affect the degree of new calcified tissue formation. To cite this article:
Beitlitum I, Artzi Z, Nemcovsky CE.
Clinical evaluation of particulate allogeneic with and without autogenous bone grafts and resorbable collagen membranes for bone augmentation of atrophic alveolar ridges.
Clin. Oral Impl. Res. 21 , 2010; 1242–1250.
doi: 10.1111/j.1600‐0501.2010.01936.x     

Parietal bone as graft material for maxillary sinus floor elevation: structure and remodeling of the donor and of recipient sites

Particulate parietal bone is used for maxillary sinus floor elevation procedure prior to dental implant placement. However, data on internal structure of the parietal bone and on graft remodeling and incorporation in the host bone are limited. We determined the structure and remodeling activities of 24 parietal bone specimens sampled at time of sinus grafting (T1 samples), and the amount and turnover of bone formed at the recipient site at time of implant placement (T2 samples, obtained 10 months after T1 samples, on average). In T1 samples, the outer cortex was 1.16+/-0.45 mm thick, had a typical haversian structure, and showed a low level of remodeling. In the cancellous portion of the samples, trabecular bone volume represented 52.8+/-10.3%. Bone remodeling was more active in the cancellous portion than in the cortical portion, but few osteoblasts and osteoclasts were seen. T2 samples consisted solely of trabecular bone, which occupied 49.4+/-18.4% of total sample volume. The boundary between new bone and the recipient bed was not discernible. Remnants of the graft particles were embedded within new bone, and showed signs of intense resorption. Bone remodeling was highly active, as shown by the presence of numerous osteoclasts resorbing new bone, together with thick osteoid seams and large osteoblasts. A loose cotton-like mineralized material was frequently observed in the marrow spaces; this acellular and non-collagenous material was strongly stained by toluidine blue, suggesting a glycoprotein nature. This study offers insights into cortical and trabecular bone structure and shows the low-level remodeling activity of parietal bone. About 10 months after grafting, the grafted chips were incorporated in new bone and almost completely resorbed. This high turnover may be beneficial for implant placement.     

重组人骨形成蛋白-2聚氰基丙烯酸正丁酯纳米微球缓释系统对骨髓间充质干细胞增殖分化影响的实验研究

目的明确重组人骨形成蛋白(rhBMP)-2聚氰基丙烯酸正丁酯纳米微球缓释系统对骨髓间充质干细胞(BMSCs)增殖和分化的影响。方法在rhBMP-2纳米微球作用于BMSCs后,采用嗜银蛋白(AgNORs)染色法检测细胞的增殖状况;采用反转录PCR方法,观察细胞中骨钙素、碱性磷酸酶及Ⅰ型胶原mRNA水平表达的改变以反映对细胞分化的影响,并与单纯rhBMP-2作用细胞后的结果进行比较。结果该纳米微球缓释系统能显著促进BMSCs的增殖以及细胞中骨钙素、碱性磷酸酶及Ⅰ型胶原mRNA水平的表达,且均高于单纯rhBMP-2的作用。结论rhBMP-2纳米微球缓释系统促进BMSCs增殖以及向成骨细胞方向分化的作用强于单纯rhBMP-2的作用。     

A feasibility study evaluating an in situ formed synthetic biodegradable membrane for guided bone regeneration in dogs

Purpose: The aim was (1) to evaluate the soft‐tissue reaction of a synthetic polyethylene glycol (PEG) hydrogel used as a barrier membrane for guided bone regeneration (GBR) compared with a collagen membrane and (2) to test whether or not the application of this in situ formed membrane will result in a similar amount of bone regeneration as the use of a collagen membrane. Material and methods: Tooth extraction and preparation of osseous defects were performed in the mandibles of 11 beagle dogs. After 3 months, 44 cylindrical implants were placed within healed dehiscence‐type bone defects resulting in approximately 6 mm exposed implant surface. The following four treatment modalities were randomly allocated: PEG+autogenous bone chips, PEG+hydroxyapatite (HA)/tricalcium phosphate (TCP) granules, bioresorbable collagen membrane+autogenous bone chips and autogenous bone chips without a membrane. After 2 and 6 months, six and five dogs were sacrificed, respectively. A semi‐quantitative evaluation of the local tolerance and a histomorphometric analysis were performed. For statistical analysis, repeated measures analysis of variance (ANOVA) and subsequent pairwise Student's t‐test were applied (P<0.05). Results: No local adverse effects in association with the PEG compared with the collagen membrane was observed clinically and histologically at any time‐point. Healing was uneventful and all implants were histologically integrated. Four out of 22 PEG membrane sites revealed a soft‐tissue dehiscence after 1–2 weeks that subsequently healed uneventful. Histomorphometric measurement of the vertical bone gain showed after 2 months values between 31% and 45% and after 6 months between 31% and 38%. Bone‐to‐implant contact (BIC) within the former defect area was similarly high in all groups ranging from 71% to 82% after 2 months and 49% to 91% after 6 months. However, with regard to all evaluated parameters, the PEG and the collagen membranes did not show any statistically significant difference compared with sites treated with autogenous bone without a membrane. Conclusion: The in situ forming synthetic membrane made of PEG was safely used in the present study, revealing no biologically significant abnormal soft‐tissue reaction and demonstrated similar amounts of newly formed bone for defects treated with the PEG membrane compared with defects treated with a standard collagen membrane.     

Improvement in the osteoblastic cellular response to a commercial collagen membrane and demineralized freeze-dried bone by an amino acid derivative: an in vitro study

Purpose: The objectives of this in vitro study were (1) to determine whether a commercially available collagen membrane (CM) or human demineralized freeze‐dried bone (DFDB) particles adversely affected viability or function in cultured osteoblasts through oxidative stress, and, if so, (2) to determine whether N‐acetyl cysteine (NAC) successfully prevented loss of viability and dysfunction in osteoblasts. Materials and methods: Rat calvaria‐derived osteoblasts were seeded onto polystyrene and commercially available CM (Cytoplast^®) or DFDB (DynaGraft^?) with or without pretreatment with NAC solution. The osteoblastic response was evaluated using a flow cytometric cell viability assay, measurement of attached viable cell number, quantification of reactive oxygen species (ROS) and alkaline phosphatase (ALP) staining. Results: The percentage of viable cells on CM was <50% at 24 h after seeding. However, this increased to 70% by pretreatment with NAC. The numbers of attached osteoblasts on DFDB remained at 60% the level of that on polystyrene at 24 h after seeding, but increased to up to 90% the level of that on polystyrene with NAC pretreatment. Although collagen materials increased intracellular ROS generation 1.5–5 times that with polystyrene, this was significantly reduced by NAC pretreatment. The percentage of the ALP‐positive area was consistently 7% or less on CM and DFDB at days 7 and 14, which was restored by NAC pretreatment up to 60% or more. Conclusions: Commercially available CM and DFDB impaired osteoblastic viability and function and markedly increased intracellular ROS, indicating an oxidative stress‐mediated negative impact on osteoblasts. Pretreatment with NAC substantially alleviated these cytotoxic effects. To cite this article:
Yamada M, Kojima N, Att W, Minamikawa H, Sakurai K, Ogawa T. Improvement in the osteoblastic cellular response to a commercial collagen membrane and demineralized freeze‐dried bone by an amino acid derivative: an in vitro study.
Clin. Oral Impl. Res. 22 , 2011; 165–172.
doi: 10.1111/j.1600‐0501.2010.01975.x     

A novel bone scraper for intraoral harvesting: a device for filling small bone defects

AIM: To evaluate histologically the morphology and characteristics of bone chips harvested intraorally by Safescraper, a specially designed cortical bone collector. MATERIAL AND METHODS: Bone chips harvested near a bone defect or in other intraoral sites were grafted into a post-extractive socket or applied in procedures for maxillary sinus floor augmentation or guided bone regeneration. Core biopsies were performed at implant insertion. Undecalcified specimens embedded in PMMA were studied by histology, histochemistry and SEM. RESULTS: Intraoral harvesting by Safescraper provided a simple, clinically effective regenerative procedure with low morbidity for collecting cortical bone chips (0.9-1.7 mm in length, roughly 100 microm thick). Chips had an oblong or quadrangular shape and contained live osteocytes (mean viability: 45-72%). Bone chip grafting produced newly formed bone tissue suitable for implant insertion. Trabecular bone volume measured on biopsies decreased with time (from 45-55% to 23%). Grafted chips made up 50% or less of the calcified tissue in biopsies. Biopsies presented remodeling activities, new bone formation by apposition and live osteocytes (35% or higher). DISCUSSION AND CONCLUSIONS: In conclusion, Safescraper is capable of collecting adequate amounts of cortical bone chips from different intraoral sites. The procedure is effective for treating alveolar defects for endosseous implant insertion and provides good healing of small bone defects after grafting with bone chips. The study indicates that Safescraper is a very useful device for in-office bone harvesting procedures in routine peri-implant bone regeneration.     

犬骨髓基质干细胞向成骨细胞诱导分化及细胞片层的制备

目的分离培养犬骨髓基质干细胞（bone marrow stromal cells,BMSCs）并向成骨细胞诱导分化,制备骨髓基质干细胞的细胞片层。方法密度梯度离心法分离犬骨髓基质干细胞,接种于DMEM成骨诱导培养基,向成骨细胞诱导分化,利用温度反应性培养皿的温度感应性,制备细胞片层。观察犬BMSCs、成骨细胞诱导分化及细胞片层形态学特点与生物学特性。结果分离培养出犬BMSCs,成功地向成骨细胞实现诱导分化,并观察到钙结节。利用温度反应性培养皿的温度感应性,收获了完整的细胞片层。结论细胞片层技术与传统的骨组织工程方法相结合,将有望构建出含板层骨结构的大块组织工程骨。     

The shape of a bone scraper: an in vitro pilot study using porcine bone chips

Bone scrapers are commonly used to harvest autologous bone in oral and implant surgery. The angle of the cutting blade is a variable that distinguishes bone scrapers. In the present study, the impact of the angle of the cutting blade on the in vitro characteristics of harvested bone was determined. Bone scrapers with blade angles of 15°, 25°, 35°, 45°, and 55° were used to harvest porcine cortical mandibular bone. The number and characteristics of the cells that grew out from the bone chips were examined. The data showed that, independent of the angle of the cutting blade, viable cells were barely detectable in fresh bone grafts. However, cells with a fibroblastic morphology appeared within 1 week in the culture dishes. After 21 days, the number of cells did not differ significantly between the five preparations. Moreover, cells responded to incubation with bone morphogenetic protein 7 (BMP-7) with an increased alkaline phosphatase activity, irrespective of the preparation. The data suggest that bone scrapers with different cutting angles produce bone chips with comparable in vitro characteristics.     

An isolation and in vitro culturing method for human intraoral bone cells derived from dental implant preparation sites

In dental implantology, the biocompatibility of the osseous tissue to the implant surface and to local environmental factors plays an important role in the process of healing. Bone cells derived from intraoral osseous tissue proves to be an important source of the osteoprogenitor cells required for healing of the petiodontium around implants. Historically, the rat calvaria model has been employed to study the effects of various dental treatments on bone in Vito. However, there are morphological and functional differences which exist between bone cells derived from rat calvaria and human intraoral osseous tissue that impose certain limitations on the usefulness of the rat calvaria model for dental implant applications. Therefore, an in vitro culturing method for the isolation, growth and maintenance of human intraoral bone cell cultures derived from osseous tissues is truly warranted. In addition, a method for the accurate characterization of these bone cells as osteoblasts is also vital. The specific objective of this study was to establish isolation and in vitro culturing methods utilizing human intraoral bone cells derived from dental implant preparation sites. This paper describes techniques for the harvesting of human bone cells from the intraoral derived osseous tissues and discuss the procedures for maintaining the primary intraoral bone cell culture. In addition, our studies utilize established protocols for the characterization of these cells as osteoblasts by means of alkaline phosphatase activity determination, identification of cellular osteonectin and osteocalcin antigens, establishing the presence of cells expressing type I collagen and determining the ability of cells to produce calcifications. The utilization of intraoral osseous tissue may prove useful for future dental implant research by providing an in vitro model system more closely related to conditions encountered clinically.     

牙种植外科骨收集器内骨碎屑成骨活性的研究

目的:通过组织学、体外细胞培养和自体移植研究收集骨的成骨活性并探讨其作为骨组织工程中种子细胞来源的可能性。方法:2003年10月至2005年10月,应用收集器收集种植窝制备时钻出的骨碎屑10例,取样本部分骨屑脱钙后行组织学观察;样本剩余部分应用组织贴块法行体外培养,观察细胞的游出、增殖和传代情况,应用碱性磷酸酶染色和连续培养矿化结节染色进行鉴定,并行扫描电镜观察。选择种植体颈部骨缺损7处,应用收集骨即刻移植修复,二期手术时观察移植物成活情况并刮取少许行组织学检查。结果:光镜下观察收集物以骨组织为主,10例样本中8例成功培养出成骨样细胞,碱性磷酸酶阳性染色率超过70%,体外连续培养茜素红染色可见桔红色结节,扫描电镜可见细胞贴附于纳米羟基磷灰石并活跃生长。牙种植二期手术时原缺损处已覆盖成熟骨质,组织学显示为密质骨。结论:收集骨具有良好的成骨活性,可用于修复种植体周围小的骨缺损,也可作为骨组织工程中种子细胞的来源。     

雌激素对大鼠骨髓基质细胞增殖和分化的影响

目的：研究雌激素对大鼠骨髓基质细胞增殖和分化的影响,探讨其对成骨的影响。方法：体外培养4周龄雌性大鼠骨髓基质细胞,以不同浓度雌激素作用于成骨细胞,用噻唑蓝法测定细胞增殖情况;对硝基酚磷酸酯法测定细胞碱性磷酸酶活性;检测培养液中羟脯氨酸含量,了解细胞Ⅰ型胶原的分泌情况;通过矿化结节计数反映细胞的矿化能力。结果：雌激素促进成骨细胞增殖,增加碱性磷酸酶活性;提高培养液中羟脯氨酸含量,峰值浓度为10^-7mol／1,与对照组比较有统计学意义（P〈0．05）;雌激素显著提高细胞的矿化能力,10^-7mol／L浓度作用最显著（＋45．4％,P〈0．05）。结论：雌激素促进成骨细胞增殖和分化,提高其矿化能力,从而刺激骨形成。     

Insulin promotes bone formation in augmented maxillary sinus in diabetic rabbits

The role of insulin during the formation of bone in the augmented space of the maxillary sinus in patients with diabetes is unclear. The authors compared the differences in bone formation after maxillary sinus floor elevation in diabetic and healthy animals and evaluated the effects of insulin on osteogenesis and the differentiation and activities of the osteoblasts. 10 male Japanese white rabbits were divided into two groups after diabetic induction by a single injection of monohydrated alloxan and having maintained steady blood glucose levels. The groups included the diabetes mellitus group (DM; n=5) and the DM+insulin group (n=5); another five healthy rabbits comprised the control group. Maxillary sinus floor elevation was performed by grafting hydroxyapatite particles. Compared with the control group, the newly formed bone area, number of blood vessels and osteoblasts, collagen I content and serum osteocalcin levels were significantly decreased in DM rabbits (P<0.01). Insulin treatment reversed the decrease in bone formation, blood vessels, osteoblasts, collagen I and serum osteocalcin (P<0.01). Insulin treatment also promoted osteogenesis in the augmented space of the diabetic rabbits, which might have resulted from promotion of osteoblast differentiation and upregulation of neovascularization.     

Lipopolysaccharide alters decorin and biglycan synthesis in rat alveolar bone osteoblasts: consequences for bone repair during periodontal disease

A prime pathogenic agent associated with periodontitis is lipopolysaccharide (LPS) derived from Porphyromonas gingivalis . This study investigated the effects of P. gingivalis LPS on osteoblasts, which are responsible for alveolar bone repair. Bone cells were obtained from explants of rat alveolar bone chips and cultured with 0–200 ng ml⁻¹ of P. gingivalis LPS. Porphyromonas gingivalis LPS significantly increased cell proliferation and inhibited osteoblast differentiation, as judged by reduced alkaline phosphatase activity. Analysis of biglycan mRNA and protein levels indicated that P. gingivalis LPS significantly delayed the normally high expression of biglycan during the early stages of culture, which are associated with cell proliferation and early differentiation of progenitor cells. In the presence of P. gingivalis LPS, decorin expression by the alveolar bone cells was reduced during periods of culture relating to collagen fibrillogenesis and mineral deposition. Analysis of glycosaminoglycan chains conjugated to these proteoglycans suggested that in the presence of P. gingivalis LPS, dermatan sulfate persisted within the matrix. This study suggests that P.  gingivalis LPS influences the expression and processing of decorin and biglycan in the matrix, altering alveolar bone cell activity and osteoblast phenotype development. The consequences of this altered expression in relation to hindering bone repair as part of the cycle of events during periodontal disease are discussed.     

Local biochemical markers of bone turnover: relationship to subsequent density of healing alveolar bone defects

OBJECTIVES: This pilot study was designed to test whether biochemical markers of bone turnover in washes of periosteal or trabecular alveolar bone surfaces could be correlated with increases in bone density of an adjacent healing implant socket. METHODS: Ten subjects had a canula inserted into the alveolar crest and sterile phosphate-buffered saline was washed over the periosteal and trabecular surfaces and collected. Surgical flaps were reflected, 5 mm diameter bone cores were removed from the bone wash site, and standardized radiographs were taken. The sites were allowed to heal for 12 weeks, and radiographs were repeated. Bone washes of the healing sites were also collected after 2 and 12 weeks. Washes were analysed for bone turnover markers osteocalcin (OC; radioimmunoassay) and C-terminal telopeptide of Type 1 collagen (ICTP; enzyme-linked immunosorbent assay (ELISA)), and blood component albumin (ALB; ELISA). Changes in bone density during healing were determined by radiographic absorptiometry. RESULTS: OC/ALB and ICTP/ALB ratios were higher for trabecular than periosteal washes at baseline (p相似文献