Background A practical problem impeding clinical translation is the limited bone formation seen in artificial bone grafts. Low-pressure/vacuum seeding and dynamic culturing in bioreactors have led to a greater penetration into the scaffolds, enhanced production of bone marrow cells, and improved tissue-engineered bone formation. The goal of this study was to promote more extensive bone formation in the composites of porous ceramics and bone marrow stromal cells (BMSCs). Methods BMSCs/β-tricalcium phosphate (β-TCP) composites were subcultured for 2 weeks and then subcutaneously implanted into syngeneic rats that were split into a low-intensity pulsed ultrasound (LIPUS) treatment group and a control group. These implants were harvested at 5, 10, 25, and 50 days after implantation. The samples were then biomechanically tested and analyzed for alkaline phosphate (ALP) activity and osteocalcin (OCN) content and were also observed by light microscopy. Results The levels of ALP activity and OCN content in the composites were significantly higher in the LIPUS group than in the control group. Histomorphometric analysis revealed a greater degree of soft tissue repair, increased blood flow, better angiogenesis, and more extensive bone formation in the LIPUS groups than in the controls. No significant difference in the compressive strength was found between the two groups. Conclusion LIPUS treatment appears to enhance bone formation and angiogenesis in the BMSCs/β-TCP composites. Chin Med J 2014;127 (4): 669-674

目的 探讨对重症肱骨外上髁炎更有效的局部注射方法.方法 80 例重症肱骨外上髁炎病例经局部封闭注射治疗,效果不满意的患者通过Peppering technique 注射法(Pep 法)或常规封闭注射方法追加治疗,经过12 个月观察临床疗效,通过Nirschl 评分法评估比较不同注射方法的治疗效果.结果 80 例肱骨外上髁炎患者经初期局部封闭治疗,优良率为47.5%;经追加治疗后最终优良率为80.0%.两种追加治疗方法临床效果比较,Pep 法优于常规封闭注射方法(P ＜0.01).对于局部封闭追加治疗效果不满意病例,再次采用Pep 法注射仍能取得较好疗效.结论 肱骨外上髁炎的局部封闭治疗是一种简单有效的治疗方法,而对于难治性肱骨外上髁炎病例,Pep 法追加治疗是一个更有效的临床选择.

目的:多孔生物材料中心部所存在的气泡有可能限制了细胞和培养液进入中心部,从而限制了材料内细胞的进一步增殖和分化,继而影响移植后的体内骨形成.实验设计使用低压方法排除多孔材料生物材料β-TCP内的气泡,检测细胞/β-TCP复合体的成骨能力,以验证其可行性.方法:实验于2003-04/2005-06在东京医科齿科大学医学部骨科实验室完成.在体外培养扩增骨髓间充质细胞后,将细胞置于成骨分化的培养液中,通过常压(大气压,101.1 kPa)和低压(13.3 kPa)两种方法将细胞灌注种植入72个多孔β-TCP人工骨中.细胞种植后检测β-TCP人工骨内含有的细胞悬浊液量,细胞生存率和细胞种植效率;应用扫描电镜观察细胞种植后人工骨内细胞附着情况.并在继续细胞分化培养1,2,3,4周时检测人工骨的DNA含量和碱性磷酸酶活性.结果:①低压组人工骨的细胞悬浊液量、全细胞种植效率和活细胞种植效率均高于常压组(P＜0.01);两种方法细胞种植后的细胞生存率没有明显不同(P＞0.05).②扫描电镜观察显示低压组人工骨内附着更多的骨髓间充质细胞.③低压组细胞分化培养过程中不同时点的人工骨碱性磷酸酶活性和DNA含量均高于常压组(P＜0.01).结论:13.3 kPa低压细胞灌注法可以明显提高多孔人工骨的细胞种植和成骨能力.低压细胞种植法是一个既简单方便又实用有效的方法.

Although human bone marrow stromal cells (MSCs) have been used for clinical bone reconstruction, how the physiological status of patients and culture conditions of MSCs affect the result of bone reconstruction must be clarified to use MSCs in a clinical stage. If in vitro parameters of the status of MSCs may be correlate with in vivo bone formation capability, the better cells for clinical bone reconstruction can be defined by the parameters. In order to explore the parameters and define the optimum cells for clinical use, the proliferation and differentiation capabilities in vitro and the in vivo bone formation capability of MSCs were analyzed. An age-related proliferation capability was found. The in vitro alkaline phosphatase activity of bone formation finding groups was higher than that of the no bone formation group. This may be provide a parameter to obtain the optimum cells for clinical use to benefit improving the cure efficiency. In this study, it is preferable that MSCs of passage 1 have stronger osteogenic potential than those of passage 2 and 3 in vitro, and might be suitable for clinical application to bone tissue engineering.

Macaque lumber posterolateral spine fusion (PLF) was performed by using beta-TCP graft combined with bone marrow derived stromal cells (MSCs), to evaluate whether a beta-TCP/MSCs hybrid can be used for PLF instead of autogenous bone graft. Nine crab-eating macaque underwent bilateral PLF at L4-L5. The implants were divided into three groups: 1) beta-TCP/MSCs hybrid, 2) autogenous bone, and 3) beta-TCP. Six monkeys were sacrified at 12 weeks and three monkeys were sacrificed at 24 weeks after implantation. Manual palpation, radiography, micro computed tomography, peripheral quantitative computed tomography (pQCT), and histology were used to assess bone formation. Manual palpation and X-ray showed that 83.3% of hybrid groups and 66.7% of autogenous groups achieved solid spine fusion, whereas none of other groups fused. Histological analysis showed that all of the hybrid groups achieved massive bone formation. Bone mineral density (BMD) evaluated with pQCT in the hybrid groups increased by additional new bone. Beta-TCP/MSCs hybrid can be used for PLF instead of autogenous bone graft. Thus it can be hypothesized that the monkey PLF can simulate human PLF.

Recently, hydroxyapatite/collagen (HAp/Col) nanocomposites incorporated into bone remodeling processes have been developed. Because bone tissue reaction to the composites is obviously suitable for bone tissue engineering, a novel HAp/Col–alginate hybrid material, as one variety of the HAp/Col implants, was prepared to expand the limited applications of the composite. The ability of HAp/Col–alginate as a bone filler was investigated in the rat femur and as a carrier of BMP using an ectopic bone formation model. Active bone formation around the material and tissue invasion into the material were observed throughout the experiment in bone, compared to those observed for simple alginate and porous HA. Furthermore, HAp/Col–alginate was investigated as a carrier of recombinant human bone morphogenetic protein 2 (rh-BMP2). The HAp/Col–alginate (20 μl) with the rh-BMP2 (100 μg/ml, 15 μl) showed bone formation throughout the implant 5 weeks after implantation without obvious deformation of the material, whereas bone formation was observed only in a part of a squashed collagen sponge.