| Background and objectiveAvascular necrosis of femoral head (ANFH) is a common orthopedic disease, which is specially destructive to hip joint. As the pathogenesis of ANFH has not been clarified, it is still difficult to treat. Patients of later stage ANFH always need total hip replacement, early treatment is important. Therefore, the question of how to treat early stage ANFH, in order to prevent the progression of the disease and maintain the shape of femoral head has raised a lot of concern. Recent studies have shown the association between bone mesenchymal stem cells (BMSCs) and ANFH, as it is believed that the decrease of BMSCs attributs to insufficient reconstruction of femoral head.More and more studies has reported that treatment of early stage ANFH by core decompression(CD) combined with BMSCs transplant. Currently most treatments extract autologus bone marrow of patients to obtain stem cells and inject the cells directly into the necrotic area after simple separation. However, the method is unable to produce enough amount of stem cells in the necrotic areas which greatly affected the outcome of the treatment. Yet the method of in vitro culturing using xenogeneic serum has the risk of potential antigenicity and pathogenicity, which greatly limits the clinical application. The new treatment of ANFH should solve the following key problems: How to realize fast expansion of human mesenchymal stem cells (hMSCs) in vitro; how to improve adhesion efficiency; how to avoid the immune response caused by allogeneic or xenogeneic serum. Considering all the problem above, We will treat early stage ANFH by core decompression combined with BMSCs implantation and investigated their efficacy and safety. MethodsThe experiment includes two partsPart 1.The human bone mesenchymal stem cells culture using autologous serum in vitro.1. Human bone mesenchymal stem cells was cultured using autologous serum in vitro1.1 General data:10 patients of early stage ANFH from Southwest Hospital were included. In this study, all patients were informed consent. There 9 males and 1 female with an average age of 36.2 years (19-45 years).The causative factors were long corticosteroids-use in 2 cases, chronic alcohol exposure in 3 cases and no obvious predisposing causes in 5 cases. The mean disease course was 1.1 years (4 months to 2 years).1.2 Preparation of autologous serum, BMSCs was analyzed with immunohistochemistry.200ml of peripheral venous blood of the patients were collected and separated by the Department of Blood Transfusion, to obtain 100ml of autologous serum(AS). BMSCs were extracted from the bone marrow blood, cultured and expanded in vitro with autologous serum. The growth and morphologic change of BMSCs were observed under inverted light microscope. The cell proliferation and growth of primary culture and serial subcultivation were observed. The purity of cells was analyzed with immunohistochemistry.2. The influence of different factors on the proliferation of BMSCs2.1 According to the factors, the patients were separated into 3 groups: steroid group(2), alcohol group(3), idiopathic group(5);2.2 Influence of different concentration on the proliferation of BMSCs:Group A (10% fetal bovine serum), group B (5% autologous serum), group C (10% autologous serum), group D (15% autologous serum), and group E (20% autologous serum);2.3 In vivo proliferation of BMSCsThe same method above2.4 Observation parameters:Morphologic changes of cells, serial subcultivation cell population doubling time(PDT);2.5 Statistic method:The resuets were analyed by T-test. Part 2. Treatment of early avascular necrosis of femoral head by core decompression combined with bone mesenchymal stem cells transplant.1.General dataFrom May 2006 to October 2008, 8 patients with early ANFH treated. Of 8 cases (16 hips), there 7 males and 1 female with an average age of 35.7 years (19-43 years), including 6 hips of stage IIa, 4 hips of stage IIb, 4 hips of stage IIc and 2 hips of stage IIIa according to the system of the Association Research Circulation Osseous. The causative factors were long corticosteroids-use in 2 cases, chronic alcohol exposure in 3 cases and no obvious predisposing causes in 3 cases. The mean disease course was 1.1 years (4 months to 2 years). The patients were randomly divided into two groups according to left or right side: Group A (control group), only the core decompression was used; Group B (experiment group), both the core decompression and implantation of autologous BMSCs were used.2.Surgical procedureCore decompression of femoral head: with the help of intra-operative X-rays, three tunnels (diameter of 5.0mm) were drilled from greater femoral tuberosity to the necrosis area below the cartilage of femoral head.Transplantation of BMSCs: extract and culture autologous BMSCs in vitro, co-culture P3 BMSCs with DBM, fill the tunnels with the above DBM compound.3. Peroperative periodCommunication with the patients were done by specialists concerning on the matters of all possible complications and post-operative rehabilitation.4. Evaluation of effectivit and securityThe Harris hip score and visual analogue scale (VAS) score for pain were determined, imaging evaluation was carried out by X-rays and MRI pre-operatively and post-operatively. The erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), liver function, renal function, IgM, IgG and IgA were detected for safety evaluation.This clinical research was approved by the Ethics Committee of Southwest Hospital, Third Military Medical University.Results1. BMSCs of primary culture the cell was fusiform shape.There was adherence cell after 24 hours of primary culture.10 to 16 days, 90% cell fused. BMSCs presented distinctive vortex growth and arranged regularly. Serial subcultivation cell grew faster than the primary culture cells . On days 6 and 8, above 90% cells were fused. Different group were able to produce enough amount of bone marrow mononuclear cell after 3 generation of proliferation. Surface markers of the cells were CD105+, CD34-.2. The amounts of bone marrow mononuclear cell and expanded BMSCs were different, the amount of the idiopathic group and the alcohol group were more than the steroid group with significant difference(P<0.05); primary culture and serial subcultivation were not finished in 5% autologous serum group and 20% autologous serum group. PDT of serial subcultivation and the primary culture time of different groups of same cause: no significant difference among 10% autologous serum group, 15% autologous serum group, and 10% fetal bovine serum group (P>0.05); PDT of serial subcultivation and the primary culture time of steroid group between idiopathic group as well as steroid group and alcoholic group were significantly different, yet there is no significant difference between the idiopathic and alcoholic group.3. All incisions healed by first intention. Eight patients were followed up 12-42 months (mean is 23.5 months). The clinical symptoms of pain and claudication were gradually improved. The Harris hip scores and the VAS scores of all patients were increased significantly at 3, 6 and 12 months after surgery (P < 0.05). There was no statistically significant difference between group A and group B at 3 and 6 months after surgery (P > 0.05), but there was statistically significant difference between group A and group B at 12 months after surgery (P < 0.05). Group B was better significantly than group A. The post-operative X-rays and MRI showed that there were some new bone formation in group B. Furthermore, the necrosis area of femoral head obviously decreased in group B. In group A, femur head collapsed 12 months after surgery in 1 stage-IIIa case.4. No complication of fever, local infection occurred.Conclusion1. High purity and active proliferation BMSCs are cultured by autologous serum substitution bovine serum, which grew prosperously and maintained its biological characters for at least 3 passages. Number of cells may suffice clinical treatment needs.2. In vivo expansion activity of BMSCs of the idiopathic group and the alcohol group were more than the steroid group with significant difference; Thene was no significant difference between idiopathic group and the alcohol group;The ability of expansion of group was no significant difference among 10% autologous serum group, 15% autologous serum group, and 10% fetal bovine serum group. It is the best choice to choose the 10% autologous serum for ANFH.3. The core decompression and the core decompression combined with implant of BMSCs are both effective for the treatment of early ANFH. The core decompression combined with implantation of BMSCs is better than the core decompression alone in the relief of pain and postponing head collapse. The core decompression combined with implant of BMSCs is a safe procedure without any complication.4. Outcome of the combined treatment of BMSCs transplantation with core decompression is better than the core decompression alone in the respect of pain relief and preventing head collapsing . |
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