复合骨髓间充质干细胞的生物型异种韧带移植修复损伤前交叉韧带

背景：异种韧带易于获得,具有韧带支架结构,有利于组织长入和爬行替代,经处理可完全消除抗原性,不引起免疫排斥反应,具有良好生长支架功能 。 目的：探索生物型异种韧带替代同种异体韧带移植重建山羊前交叉韧带损伤的可行性。 方法：将24只健康山羊随机等分成A,B,C三组,取羊的左膝关节,制作成前交叉韧带断裂缺失模型,建立胫骨、股骨端骨隧道后,A,B,C组分别植入复合骨髓间充质干细胞的生物型异种韧带、生物型异种韧带和同种异体韧带。 结果与结论：复合骨髓间充质干细胞的生物型异种韧带植入山羊体内后,无明显排斥反应,具有良好的组织相容性,植入韧带作为功能性支架重建前交叉韧带,在动物膝关节内环境的诱导下,使自体新生组织长入并替代支架,形成自体新生韧带,骨腱部愈合良好,但其与同种异体韧带重建前交叉韧带在组织学、免疫反应、生物力学方面差异无显著性意义,而植入复合骨髓间充质干细胞的生物型异种韧带山羊宿主自体组织可以长入并建立微循环。提示复合骨髓间充质干细胞的生物型异种韧带可加速微循环建立,促进韧带生长,尤其对韧带再血管化作用显著,但对韧带生物力学无明显影响。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

骨组织工程的种子细胞--骨髓基质细胞的研究进展

骨髓基质细胞作为骨组织工程的种子细胞具有广阔前景.许多实验证实骨髓基质细胞具有间充质干细胞特性,表现为较强的增殖能力和向多种间充质细胞分化的潜能.目前已建立了体外培养骨髓基质干细胞的方法,而且正在摸索进一步纯化的方法和诱导分化的条件.已有利用其成骨特性体内移植实验,表明在适当的条件下,接种在组织工程材料上的骨髓基质细胞可以形成新骨.     

Development of self-assembled, tissue-engineered ligament from bone marrow stromal cells

The human anterior cruciate ligament is ruptured 200,000 times per year in the United States, resulting in medical costs of $1 billion. The standard treatment is patellar tendon autograft, but this treatment is suboptimal because of lengthy recovery time, arthritis, donor site morbidity, and degenerative joint disease. This study aimed to engineer scaffold-free ligament analogs from a clinically relevant cell source and to examine mechanical and histological properties of the resulting engineered tissue. Porcine bone marrow stromal cells were seeded on laminin-coated substrates with silk suture segments as anchor points. Cells developed into monolayers that subsequently delaminated and self-organized into cohesive rod-like tissues that were held in tension above the substrate. After 14 days of maturation, scanning electron microscopy revealed a well-organized extracellular matrix, aligned collagen fibers, and a collagen fibril diameter of 51.1+/-77 nm. Histological evaluation showed that constructs were composed of approximately 60% collagen. During tensile tests to failure, constructs had a stress of 2.11 +/- 0.13 MPa, a strain of 28.8 +/- 0.95%, a force of 0.26 +/- 0.02 N, and a tangent modulus of 15.4+/-1.04 MPa. Mechanically and histologically, engineered ligament resembled native embryonic connective tissue and had an ultimate stress approximately 15% of native adult mouse tissue.     

Graft materials and bone marrow stromal cells in bone tissue engineering

Bone augmentation procedures rely on osteogenic/osteoconductive properties of bone graft material (BGM). A further improvement is represented by use of autologous bone marrow stromal cells (BMSC), expanded in vitro and seeded on BGM before implantation in the bone defect. The effect of different BGMs on BMSC osteogenic differentiation was evaluated. BMSC were cultured in vitro in the presence of different BGM (natural, synthetic, and mixed origins). Cellular morphology was analyzed with scanning electron microscopy. The capability of BMSC to differentiate was determined in vitro by alkaline phosphatase gene expression and enzyme activity at different time points (7, 14, and 28 days) and in vivo by ectopic bone formation of implanted tissue constructs in an immunodeficient murine model. BGM supports the cell adhesion and osteogenic differentiation of BMSC developing a useful tool in the bone tissue engineering.     

Human bone marrow stromal cells: In vitro expansion and differentiation for bone engineering

Stromal cells from marrow hold a great promise for bone regeneration. Even if they are already being exploited in many clinical settings, the biological basis for the source and maintenance of their proliferation/differentiation potential after in vitro isolation and expansion needs further investigation.

Most studies on osteogenic differentiation of marrow stromal cells (MSC) have been performed using bone marrow from the iliac crest. In this study, MSC were derived from spare femoral bone marrow obtained during hip replacement surgery from 20 adult donors. After in vitro isolation the cells were grown in osteogenic medium, and their proliferation and differentiation analysed during in vitro expansion. We found that MSC isolated from the femur of adult patients consistently maintain an osteogenic potential. Using biochemical signals, these cells turn to fully differentiated osteoblasts with a predictable set of molecular and phenotypic events of in vitro bone deposition. When seeded on polycaprolactone-based scaffold or surfaces, the proliferation and mineralization of femur-derived MSC were modulated by the surface chemistry/topography. Despite remarkable differences between individual colony-forming ability, alkaline phosphatase production, and mineralization ability, these cells are a potential source for bone engineering, either by direct autologous reimplantation or by ex vivo expansion and reimplantation combined to a proper scaffold.     

Mesenchymal stem cell characteristics of human anterior cruciate ligament outgrowth cells

When ruptured, the anterior cruciate ligament (ACL) of the human knee has limited regenerative potential. However, the goal of this report was to show that the cells that migrate out of the human ACL constitute a rich population of progenitor cells and we hypothesize that they display mesenchymal stem cell (MSC) characteristics when compared with adherent cells derived from bone marrow or collagenase digests from ACL. We show that ACL outgrowth cells are adherent, fibroblastic cells with a surface immunophenotype strongly positive for cluster of differentiation (CD)29, CD44, CD49c, CD73, CD90, CD97, CD105, CD146, and CD166, weakly positive for CD106 and CD14, but negative for CD11c, CD31, CD34, CD40, CD45, CD53, CD74, CD133, CD144, and CD163. Staining for STRO-1 was seen by immunohistochemistry but not flow cytometry. Under suitable culture conditions, the ACL outgrowth-derived MSCs differentiated into chondrocytes, osteoblasts, and adipocytes and showed capacity to self-renew in an in vitro assay of ligamentogenesis. MSCs derived from collagenase digests of ACL tissue and human bone marrow were analyzed in parallel and displayed similar, but not identical, properties. In situ staining of the ACL suggests that the MSCs reside both aligned with the collagenous matrix of the ligament and adjacent to small blood vessels. We conclude that the cells that emigrate from damaged ACLs are MSCs and that they have the potential to provide the basis for a superior, biological repair of this ligament.     

Porcine anterior cruciate ligament fibroblasts are similar to cells derived from the ligamentum teres, another non-healing intra-articular ligament

Porcine ligament fibroblasts were cultured from the anterior cruciate (ACL), medial collateral (MCL), and ligamentum teres (LT). There were no apparent differences in confluent cellular morphology among the ligament cell types as evaluated by phase contrast microscopy. The proliferation rate of MCL cells from 24-120 h was significantly higher (p < 0.05) than that of cells from either the LT or the ACL. MCL cells produced more collagen and less non-collagenous protein than the LT and ACL as determined by [3H]proline incorporation. This resulted in MCL cells producing a higher percentage (37%, p < 0.05) of collagen relative to total protein than either the ACL (28%) or the LT (32%). The MCL cells produced a significantly higher percentage (34.7%, p < 0.05) of type-III collagen relative to total type-I and III collagen than either the ACL (29.2%) or the LT (29.5%). The LT and MCL cells had similar and significantly greater coverage of in vitro wounds than the ACL. This study provides the first in vitro study of the LT and demonstrates that fibroblasts from the LT and ACL, two ligaments that heal poorly, have similar in vitro characteristics, with the exception of wound healing.     

Assembly of bone marrow stromal cell sheets with knitted poly (L-lactide) scaffold for engineering ligament analogs

The current cell seeding technique has several disadvantages, such as low efficiency of cell attachment to scaffolds and the limited strength of cell-gel composite adhesion to scaffold. These problems warrant further study to improve the assembly of cell to scaffold. Therefore this study aims to fabricate a bone marrow stromal cells (bMSCs) sheet and assemble it on a knitted poly (L-lactide) (PLLA) scaffold for engineering ligament analogs. bMSCs were cultured to form a cell sheet in the presence of ascorbic acid. Once a sheet of bMSCs was obtained, it was assembled onto the knitted scaffold by a wrapping technique. Then the assembled structure was held in place in a spinner flask for 4 weeks. The macromorphology, histology, and biomechanics of the grafts were evaluated. The composite of cell sheet/PLLA scaffold constructs had transformed into tissuelike ligament analogs. Immunohistochemical analysis showed that the components of the analogs were similar to that of ligament tissues, consisting primarily of collagen type I and small amount of collagen type III and tenascin. The failure force of the cell/scaffold assembly under tension (46.68+/-2.29 N) was higher than that of the scaffold group (43.58+/-2.41 N; p<0.05), but tensile stiffness of the cell/scaffold group (20.6+/-1.417 N/mm) was significantly lower than that of the scaffold group (27.6+/-1.449 N/mm; p<0.05). These data showed that the incorporation of bMSCs sheet onto the PLLA scaffold could make the analog stronger and more stretchable. Therefore the approach of assembling bMSCs sheet onto knitted PLLA scaffold is promising for producing tissuelike and functional ligament analogs under dynamic fluid situation for the purpose of anterior cruciate ligament (ACL) reconstruction.     

In vitro control of human bone marrow stromal cells for bone tissue engineering

For the clinical application of cultured human mesenchymal stem cells (MSCs), cells must have minimal contact with fetal calf serum (FCS) because it might be a potential vector for contamination by adventitious agents. The use of human plasma and serum for clinical applications also continues to give rise to considerable concerns with respect to the transmission of known and unknown human infectious agents. With the objective of clinical applications of cultured human MSCs, we tested the ability of autologous plasma, AB human serum, FCS, and artificial serum substitutes containing animal-derived proteins (Ultroser G) or vegetable-derived proteins (Prolifix S6) to permit their growth and differentiation in vitro. To conserve as much autologous plasma as possible, we attempted to mix it at decreasing concentrations with the serum substitute containing vegetable-derived mitogenic factors. Under control conditions, by day 10 all the fibroblast colony-forming units (CFU-Fs) were alkaline phosphatase (ALP) positive. However, their number and size were highly variable among donors. Better CFU-F formation was obtained with Ultroser G, and with human AB serum and autologous plasma mixed at, respectively, 5 and 1% with Prolifix S6. The effects of these mixtures on CFU-F formation demonstrate synergy, with the human serum or plasma supplying the factors that favor differentiation of MSCs while Prolifix S6 supplies the mitogenic factors. Finally, we demonstrated the possibility of controlling human MSC growth and differentiation in vitro. Notably, by means of a minimal quantity of human serum or human plasma mixed with a new serum substitute containing vegetable-derived proteins, we displayed growth and differentiation of human MSCs comparable to that obtained with FCS or serum substitutes containing animal-derived proteins. These results will have crucial significance for future applications of cultured human MSCs in bone tissue engineering.     

Comparing various off-the-shelf methods for bone tissue engineering in a large-animal ectopic implantation model: bone marrow, allogeneic bone marrow stromal cells, and platelet gel

Construction of bone grafts for regenerative medicine would highly benefit from off-the-shelf components, such as allogeneic bone marrow stromal cells (BMSCs) and blood-derived growth factors from platelet concentrate. Although allogeneic BMSCs are considered immunosuppressive, their use in transplantation studies is still cautioned. In this study, we used off-the-shelf goat allogeneic BMSCs, per-operatively aspirated bone marrow (BM) and platelet gel (PLG). Ten goats received six different hybrid constructs consisting of biphasic calcium phosphate scaffolds seeded with PLG or plasma that were mixed with BM, allogeneic BMSCs or left without cells. All constructs were implanted in the paraspinal muscles for 9 weeks. Fluorochromes were administered at 2, 3, and 5 weeks to assess onset of bone formation. Analysis revealed that the scaffolds without cells yielded small amounts of bone. Allogeneic BMSCs had a positive effect on the amount and early onset of bone formation. Fresh BM did not enhance ectopic bone formation. The PLG, which contained higher levels of transforming growth factor beta than plasma, did not result in more bone either. Fluorochrome incorporation results indicate that the presence of seeded cells in the constructs accelerates bone formation. This study shows a potential role of allogeneic BMSCs in bone tissue-engineering research.     

双骨道与三骨道前交叉韧带双束重建的生物力学比较

背景：关节镜下前交叉韧带重建可以有效地解决前交叉韧带损伤引起的膝关节不稳症状,而双骨道(胫骨单骨道-股骨单骨道)与三骨道(胫骨单骨道-股骨双骨道)前交叉韧带重建是目前较为常用的重建方式。 目的：探讨双骨道与三骨道前交叉韧带双束重建对膝关节稳定性的影响。 方法：选用8具新鲜正常成人尸体膝关节标本,分别进行双骨道与三骨道前交叉韧带双束重建,然后在MTS-809生物力学测试系统上测试膝关节在胫前加载(134 N)以及胫骨旋转加载(5 N•m内旋)下屈曲0°,15°,30°,60 °,90 °时的膝关节稳定性。 结果与结论：①胫前加载：在所测的5个角度下,两重建组的胫前位移较前交叉韧带完整组均增大,但差异无显著性意义(P > 0.05);双骨道重建组较三骨道重建组胫前位移增大,但差异无显著性意义(P > 0.05)。②旋转加载：在所测的5个角度下,前交叉韧带完整组的胫前位移最小,双骨道重建组与三骨道重建组比较,在膝关节屈曲0°,15°和90°时,差异无显著性意义(P > 0.05),在膝关节屈曲30°和60°时,三骨道重建组胫前位移小于双骨道重建组,差异有显著性意义(P < 0.05);三骨道重建组与前交叉韧带完整组比较,差异无显著性意义(P > 0.05)。提示双骨道与三骨道前交叉韧带双束重建均可促进膝关节前后及旋转稳定性的恢复,三骨道前交叉韧带双束重建与双骨道前交叉韧带双束重建相比,显示了更好恢复膝关节旋转稳定性的作用。     

Regional bone density changes in anterior cruciate ligament deficient knees: a DEXA study

Bone mineral density (BMD) loss is one of the secondary problems occurring in knee joint after injury of anterior cruciate ligament (ACL). The effect of this injury on BMDs of specific regions is not clear. The aim of this study was to investigate BMD changes in unreconstructed ACL-deficient knees with subregion analysis of dual energy X-ray absorptiometry (DEXA). Precision and reliability studies of DEXA revealed that two region of interests (ROI) in medial condyle, two ROIs in lateral femoral condyle (LFC) and one ROI in medial tibial plateau (MTP) in anteroposterior (AP) DXA view and one ROI for each of distal femur, proximal tibia and patella in lateral view had high reproducibility and reliability. Thirty-two patients with complete ACL ruptures were collected for the study and uninjured sides served as the control. All the patients were male with a mean age of 30 years. Mean duration of ACL rupture was 24 months. There were significant BMD losses in both ROIs of LFC and ROI of MTP in AP view and all three ROIs of lateral view. Greatest BMD losses in AP and lateral views were at MTP and patella respectively. There was a significant association between patellar BMD loss and duration after trauma. Bone bruises in lateral condyle might be the cause of selective involvement of LFC. Periarticular bone mineral loss in ACL-deficient knees has a predilection for the specified region of interest rather than uniform periarticular loss. This may be important for graft fixation or a factor in tunnel enlargement.     

The anterior cruciate ligament deficiency as a model of brain plasticity

This paper describes a plausible mechanism for the development of brain plastic changes due to a peripheral joint injury, such as anterior cruciate ligament (ACL) rupture. Evidence for the hypothesis is based on mainly three indications derived from the literature review: (a) the existence of two different categories of ACL patients, the copers and non-copers, presenting different features of functional deficiencies, (b) the demand of a sufficient post-traumatic time (more than 6 months) for the dysfunction development and (c) the fact that the dysfunction is not limited to the injured limb but also concerns the non-injured one. Considering the fact that ACL contains mechanoreceptors, which inform the central nervous system (CNS) about joint sense position and kinaesthesia, it can be suggested that this kind of injury might be regarded as a neurophysiological dysfunction, not being a simple musculoskeletal injury. The rupture of the ACL could lead to the cessation/depletion or differentiation of the ascending afferent pathway from the injured joint towards CNS, inducing to the joint de-afferentation and consequently CNS reorganization and joint de-efferentation. In case of presumable evidence of the proposed hypothesis, its clinical application could concern several aspects of the intervention procedures. For example, a number of clinical findings, such as the functional differences presented between two separate clinical groups of patients (copers and non-copers) could be justified or the rehabilitation strategies might have to be revised, provided that certain therapeutic components have influence on facilitating brain plastic changes that induce to beneficial functional outcomes.     

Effects of pH on human bone marrow stromal cells in vitro: implications for tissue engineering of bone

The objective of this study was to address the hypothesis that changes in extracellular pH alter collagen gene expression, collagen synthesis, and alkaline phosphatase activity in bone marrow stromal cells (BMSCs). Potential effects of pH on cell function are of particular importance for tissue engineering because considerable effort is being placed on engineering biodegradable polymers that may generate a local acidic microenvironment on degradation. Human and murine single-cell marrow suspensions were plated at a density of 2 x 10(4) cells/cm(2). After 7 days in culture, the pH of the culture medium was adjusted to one of six ranges: > or = 7.8, 7.5.-7.7, 7.2-7.4, 6.9-7.1, 6.6-6.8, or < or = 6.5. After 48 h of exposure to an altered pH, alkaline phosphatase activity and collagen synthesis decreased significantly with decreasing pH. This decrease was two-to threefold as pH decreased from 7.5 to 6.6. In contrast, alpha1(I) procollagen mRNA levels increased two- to threefold as pH was decreased. The trend in osteocalcin mRNA expression was opposite to that of collagen. Small shifts in extracellular pH led to significant changes in the ability of BMSCs to express markers of the osteoblast phenotype. These pH effects potentially relate to the microenvironment supplied by a tissue-engineering scaffold and suggest that degrading polymer scaffolds may influence the biologic activity of the cells in the immediate environment.     

Mixing conditions for cell scaffolds affect the bone formation induced by bone engineering with human bone marrow stromal cells, beta-tricalcium phosphate granules, and rhBMP-2

Bone regenerative medicine via tissue engineering is expected to be an alternative treatment for conventional autogenous bone graft, as it is less invasive. One of the best triads for bone engineering is bone marrow stromal cells, calcium phosphate ceramics, and bone morphogenetic protein (BMP). However, the optimal mixing conditions for BMP-induced osteoblasts and ceramic granules remain unclear. Therefore, we investigated the effect of the mixing conditions for cell scaffolds on the bone-forming potential. The cells were mixed with beta-tricalcium phosphate (beta-TCP) granules followed by osteoblast induction with recombinant human BMP-2 (rhBMP-2) (first mixture), or were first induced with rhBMP-2 on plastic dishes and then mixed with the beta-TCP granules (last mixture) just prior to the operation. Both the first and last mixtures were transplanted into nude mice subcutaneously, with the amount of bone formation analyzed histomorphometrically. In addition, cell numbers and alkaline phosphatase (ALP) activity before transplantation was determined in both the mixtures. In vitro analyses revealed that cell numbers were greater in the last mixture, whereas ALP activity was greater in the first mixture. In vivo analyses revealed that the first mixture was much more osteogenic than the last mixture with respect to new bone formation and osteocalcin synthesis. These data suggest that cell-scaffold mixing conditions have a significant influence on the bone-forming capacity via bone engineering and that first mixture might be the optimal condition for rhBMP-2-induction of human osteoblasts.