Abstract

BACKGROUND: Ischemic femoral head necrosis is caused by local vascular injury and blood-supply insufficiency. There exists no optimal treatment for the ischemic femoral head necrosis. Thus, the improvement of the blood supply to the femoral head seems to be a key point for the treatment.
OBJECTIVE: To verify the curative effects of autologous bone marrow mesenchymal stem cell transplantation induced vascular regeneration on the improvement of ischemic femoral head necrosis via animal experiments and clinical observations.
DESIGN: Contrast animal experiment and self-controlled clinical observation.
SETTING: Cell Therapy Center, the 463 Hospital of Chinese PLA.
MATERIALS AND PARTICIPANTS: ① Animals: Twenty Japanese white rabbits in either gender and weighing 3.0-4.0 kg were purchased from Animal Experimental Center, General Hospital of Shenyang Military Area Command of Chinese PLA. The animal experiments were coincident with the ethical standards. ② Participants: 188 patients with ischemic femoral head necrosis (335 hips) having whole following-up data were selected from Cell Therapy Center, the 463 Hospital of Chinese PLA from July 2004 to July 2007. There were 113 males and 75 females, and their ages ranged from 18 to 72 years. Diagnosis was done by using X-ray photographs, nuclide scanning, MRI and CT examinations. All patients provided the informed consent, and the study was approved by the local research ethics committee.
METHODS: ① Animal experiments: The experiment was carried out at the Animal Experiment Center, General Hospital of Shenyang Military Area Command of Chinese PLA from January to June 2004. Ischemic femoral head necrosis models were established at both hindlimbs by using liquid-nitrogen refrigeration. The right side was regarded as transplantation group and the left one as control group. Mononuclear cells extracted from bone marrow were poured in the right femoral artery, while saline was poured in the left femoral artery. ②Clinical observations: Mononuclear cells were separated from autologous bone marrow of patients with ischemic femoral head necrosis after density gradient centrifugation.
MAIN OUTCOME MEASURES: ①Four weeks later, angiogenesis at both femoral arteries was observed by arteriography by using digital subtraction anglography (DSA). Moreover, bilateral femoral head samples underwent pathological sections to observe bone regeneration and repair of femoral head 4 and 12 weeks later. ②Items including hip pain, walking distance and gait, abduction and internal rotation function changes of hip joint were observed in 3, 6, 12 and 24 months after stem cell transplantation in media femoral circum flex artery, lateral femoral circum flex artery and obturator artery. In 6 months after stem cell transplantation, angiogenesis and blood supply of femoral head were observed by using arteriography. In 6, 12 and 24 months after stem cell transplantation, morphological and ischemic changes of femoral head were observed by using CT, X-ray and MRI examinations. Harris scores were used to evaluate function of hip joint before and in 3, 6, 12 and 24 months after stem cell transplantation.
RESULTS: Animal experiment: Twenty rabbits were involved in the final analysis. ① DSA-arteriography results: In 4 weeks after transplantation, blood-supply arteries in femoral head of right hindlimb in the transplantation group were more than those in the control group. ② Pathological results: In 12 weeks after transplantation, cartilage, lamellar bone and bone trabecula in the left femoral head were repaired remarkably, but left femoral head necrosis was not improved. Clinical observations: 188 patients were involved in the final analysis. ① Improvement of symptoms: Among 188 patients, 164 (87.3%) had remission of hip pain, 147 (78.4%) had function improvement, and 150 (80.0%) had elongation of walking distance. ② Imaging changes: At 6 months after transplantation, DSA-arteriography in 12 patients demonstrated that blood-supply arteries in femoral head were increased and thickened remarkably as compared with those before transplantation, and the blood flow was rapid. At 12-24 months after transplantation, lesion of bone matrix in 24 patients was improved under the X-ray, CT and MRI examinations. ③ Harris scores of hip joint: The scores at 6, 12 and 24 months after transplantation were significantly higher than those before transplantation (t= -3.423, -6.714, -9.039, P < 0.01).
CONCLUSION: Autologous bone marrow mesenchymal stem cell transplantation can effectively improve and treat ischemic femoral head necrosis.

INTRODUCTION

Avascular necrosis of femoral head (ANFH) is a high mutilation disease. There are multiple etiological factors, and all of them can lead to blood supply disorder in femoral head[1]. There are no effective ways to treat femoral head ischemia. This study was designed to investigate the curative effects of autologous bone marrow mesenchymal stem cell transplantation induced vascular regeneration on the improvement of ischemic femoral head necrosis. In this purpose we used animal experiments and performed clinical observations.

DATA AND METHODS

Animal experiment
Materials
The experiment was carried out at the Animal Experiment Center, General Hospital of Shenyang
Military Area Command of Chinese PLA from January to June 2004. Twenty Japanese white rabbits of either gender and weighing 3.0-4.0 kg were purchased from Animal Experimental Center,
General Hospital of Shenyang Military Area

Command of Chinese PLA (certification: SYXK-Army-2002-
019). Ficoll was provided by Shanghai Huajing Biological High-technological Company Limited, and liquid nitrogen by Shenyang Xinguang Group. The animal experiments were in accordance with the ethical standards.

Methods
Model preparations: Ischemic femoral head necrosis models were established at both hindlimbs of 20 rabbits by using liquid-nitrogen refrigeration discovered by Yang et al[2]. The right side was regarded as transplantation group and the left one as control group. After anesthesia with soluble pentobarbitone, bilateral tip joints received lateral incisions under aseptic manipulation, and femoral head was exposed, dislocated and covered with rubber funnel. And then, liquid nitrogen was poured in the funnel and stayed for 3 minutes. Finally, warm water was used to rewarm and replace the femoral head, and the incisions were sutured layers by layers.
Collection and preparation of bone marrow stem cells: At 7 days after establishing ischemic femoral head necrosis models by using liquid nitrogen refrigeration, rabbits were anesthetized with soluble pentobarbitone to puncture bilateral posterior superior iliac spine. 10 mL bone marrow underwent Ficoll density gradient centrifugation to extract mononuclear cell suspension with the density of 1×1011 L-1.
Stem cell transplantation: After anesthesia, abdominal median incision was given to expose abdominal aorta. And then, canal was sent to the right femoral artery after successful puncture to temporarily block arterial blood stream of hindlimbs; meanwhile, stem cell suspension was poured in the right femoral artery. With the same procedure, saline was poured in the left femoral artery, and abdominal cavity was closed.
Observational indexes: ① Walking gait: Changes of walking gait of the experimental rabbits were observed before and after model establishment and at 2-14 days after stem cell transplantation. ② Arteriography: The experimental rabbits underwent arteriography on lower limbs by using digital subtraction anglography (DSA) in 4 weeks after stem cell transplantation. Abdominal aorta was punctured, poured with 70% cystografin and photographed successively. Density of vascular distribution was contrastively observed in femoral head of both hindlimbs. ③ Pathological sections: In 4 and 12 weeks after stem cell transplantation, 3-5 pieces of femoral head collected from both hindlimbs were fixed in 10% neutral formaldehyde for 24 hours, embedded with paraffin and cut into sections. Position of bone trabecula was observed in ten high power fields. Fifty bone lacunas were counted in each field, and ratio of blank bone lacuna was accumulated. Bilateral femoral head necrosis and repair were observed contrastively.
Statistical analysis: SPSS 12.0 software was used by the third author. Data were statistically calculated with t test.

Clinical data
Subjects
188 patients with ischemic femoral head necrosis (335 hips) having undergone bone marrow mesenchymal stem cell transplantation were selected from Cell Therapy Center, the 463 Hospital of Chinese PLA from July 2004 to July 2007. There were 113 males and 75 females, their ages ranged from 18 to 72 years, and the illness course lasted for 6 months to 14 years. Among 118 patients, 47 had traumatic femoral head necrosis, 66 had estrogenic femoral head necrosis, 54 had alcoholic femoral head necrosis, and 21 had cryptogenetic femoral head necrosis. Moreover, 73 patients also had hip and knee pain, lameness and joint functional disorder. Final diagnosis was done by using X-ray, nuclide scanning, MRI and CT examinations. There were 147 bilateral femoral head necrosis and 41 lateral femoral head necrosis, a total of 335 hips. All 188 patients received Chinese herb and physiotherapy. For example, 37 patients underwent interventional thrombolysis, and 25 underwent bone-grafting operation. However, symptoms of all patients were not relieved but deteriorated continuously. According to necrosis phase criteria set by Association Research Circulation Osseous (ARCO)[2], there were 43 hips (12.9%) in the phase Ⅰ, 154 (46.0%) in the phase Ⅱ, 127 (37.8%) in the phase Ⅲ and 11 (3.3%) in the phase Ⅳ. Inclusion criteria: Patients received various treatments for more than 6 months before stem cell transplantation. The clinical symptoms were not improved but progressively deteriorated as assessed by imaging examination. Final diagnosis was made by X-ray, nuclide scanning, MRI and CT examinations. The patients were in both sexes and their age was above 18 years old. Informed consent was obtained from all patients. Exclusion criteria: Patients not desiring to be included in the study or had tumor and severe cardiocerebral diseases were excluded. The study was approved by the local research ethics committee.

Methods
Observational ways: Hip pain, walking distance, hip joint function, blood-supply arteriography, X-ray film, morphological changes of CT and MRI examinations were assessed at 3, 6, 12 and 24 months after transplantation; if the symptoms were improved, stem cell transplantation was considered as effective.
Collection and separation of bone marrow stem cells: After local anesthesia, 200-400 mL of bone marrow wereas extracted from bilateral posterior superior iliac spine under aseptic manipulation and given density gradient centrifugation to separate mononuclear cells with the density of (2.0-6.2) ×109. And then, (2.76±0.58)% CD34+ cells and (1.88±0.45)% CD133+ cells were added to prepare suspension (10-20 mL).
Blood-supply arteriography of the femoral head by using DSA: All patients underwent DSA examination and blood-supply of femoral head was evaluated at the same time. Canals were punctured to femoral artery and internal iliac artery, and then contrast medium was poured in artery by using high-pressure injection pump to observe degrees of vascular occlusion. The results demonstrated that stenosis or occlusion of media femoral circum flex artery, lateral femoral circum flex artery and obturator artery occurred in 55 hips, bilateral stenosis or occlusion in 156 hips, lateral stenosis or occlusion in 93 hips, whereas 31 hips were normal.
Stem cell transplantation: Canals were inserted into media femoral circum flex artery, lateral femoral circum flex artery and obturator artery, and then BMSC suspension was slowly perfused into arteries. If lesions occurred in the bilateral femoral head, the stem cell transplantation should be performed on bilateral media femoral circum flex artery, lateral femoral circum flex artery and obturator artery. Moreover, patients lied in bed for 12 hours after operation.
Clinical observations: ①Clinical markers: Degrees, characteristics and duration of hip pain, changes of walking distance and gait, and abduction and internal rotation function changes of hip joint were evaluated 3, 6, 12 and 24 months after stem cell transplantation. ②DSA/arteriography imaging and hip imaging indexes: At 6 months after stem cell transplantation, angiogenesis and blood supply of femoral head were observed by using arteriography. At 6, 12 and 24 months after stem cell transplantation, morphological and ischemic changes of femoral head were assessed by using CT, X-ray and MRI examinations. Harris scores including pain and joint function were used to evaluate function of hip joint before and at 3, 6, 12 and 24 months after stem cell transplantation. There were 100 points in total. The higher the scores were, the better the hip joint function was.
Statistical analysis: The statistical analysis of clinical data was as the same as that of the animals.

RESULTS

Animal experiments
Quantitative analysis of the experimental animals
Twenty rabbits were involved in the final analysis.

Walking gait
After femoral head necrosis modeling, the experimental rabbits were not able to stand with both hindlimbs. In 4 weeks after stem cell transplantation, standing ability and activity of the transplanted right hindlimb were improved remarkably, but there were no obvious changes on the control limbs.

Arteriography
In 4 weeks after transplantation, the experimental rabbits underwent arteriography on the bilateral femoral head, and the results indicated that blood-supply arteries of the transplanted right hindlimb were increased and thickened in the transplantation group as compared with that in the control group. In addition, the blood flow was faster.

Pathological sections
Morphological changes of bilateral femoral head were observed in 4 and 12 weeks after transplantation. In the 4th week, obvious necrosis was observed in the bone tissues of bilateral femoral head. Bone trabecula was thin and broken; the distance was enlarged; osteocytes were reduced; pyknosis and deep staining were found; blank bone lacuna was increased; cartilage was in necrosis. In the 12th week, breakage of bone trabecula was relieved at transplanted side; arrangement was in a good order; blank bone lacuna was decreased; osteoblasts were in one line at the surface of bone trabecula; newborn bone was generated; blood cells were increased in medullary cavity; remarkable blood capillary and interstitial cell hyperplasia were observed (Figure 1). Degeneration of cartilage at the transplanted side was relieved as compared with that at the control side.

Clinical observations
Changes of clinical symptoms
Follow-up lasted for 3 to 24 months (mean time of 11.2 months). For instance, follow-up lasted for 3 months in 188 cases, for 6 months in 117 cases, for 12 months in 76 cases and for 24 months in 45 cases. Among 188 patients, 164 (87.3%) had remission of hip pain, 147 (78.4%) had function improvement, and 150 (80.0%) had elongation of walking distance (Table 1).

Imaging observation
At 6 months after transplantation, DSA-arteriography in 12 patients demonstrated that blood-supply arteries in femoral head of 12 cases were increased and thickened remarkably as compared with those before transplantation, and the blood flow was rapid (Figure 2). In 12-24 months after transplantation, lesion of bone matrix in 24 patients was improved under the X-ray, CT and MRI examinations. Moreover, lesion area of bladder was small; fracture line showed a tendency to healing; incisure of cartilage at the surface of femoral head disappeared; hip joint surface was smooth (Table 2).

Harris scores of hip joint
The scores at 6, 12 and 24 months after transplantation were significantly higher than those before transplantation (P < 0.01). The longer the time from the stem cell transplantation was, the higher the Harris scores were (Table 3).

DISCUSSION

Ischemic femoral head necrosis of the adults is caused by local nourishing vascular injury and blood-supply insufficiency. In addition, it can further induce ischemia, degeneration and necrosis of bone matrix, breakage of bone trabecula, collapse of femoral head and functional disorder of hip joint[3]. Therefore, the improvement of the blood supply of femoral head becomes a key point for the treatment. Autologous bone marrow mesenchymal stem cell transplantation induced vascular regeneration has become a novel method to treat various ischemic diseases. Stem cells have multi-directional differentiation; moreover, they can directionally differentiate into other cells under the influence of various cytokines into a specific environment and promote repair of injured tissues[4]. In the study of Gehling et al[5-6], CD133+ cells derived from peripheral blood can differentiate into endothelial cells in vivo and promote the repair of blood stream in ischemic limbs. These precursors are considered to present high proliferation and differentiation potency, and they participate in ischemic local compensative revascularization by inducing angiogenesis. Animal experiments demonstrated that, after the transplantation into ischemic myocardium or limbs, autologous bone marrow, peripheral stem cells and endothelial progenitor cells (EPC) might form new blood capillaries in local regions[7]. Yang et al[8] achieved notable effects of autologous peripheral stem cell transplantation in the treatment of various vascular diseases of ischemic lower limbs, and the results indicated that stem cells could differentiate into newborn vessels under hypoxic condition, effectively perfuse ischemic tissues, inhibit necrosis of tissues and improve ischemic symptoms. The researches about arterial perfusion of bone marrow stem cells into necrotic femoral head of rabbits demonstrated that bone marrow stem cells could dredge vessels in injured femoral head, improve necrosis of femoral head and blood circulation of peripheral tissues and promote vascular regeneration[9]. The results in this study suggested that vascular regeneration was observed in femoral head in 1 month after arterial stem cell transplantation; while, regeneration of cartilage and lamellar bone were observed in necrotic region in 3 months after transplantation.
There were no notable effects of various therapies except autologous bone marrow mesenchymal stem cell transplantation on 188 patients with ischemic femoral head necrosis, because ischemia in femoral head region was not remarkably improved. Under local hypoxic conditions, stem cells could differentiate into endothelial cells by using arterial stem cell transplantation, and the newborn vessels could improve blood supply of femoral head. DSA-arteriography in 12 patients demonstrated that a lot of vessels were generated in femoral head at 6 months after transplantation; blood flow was rapid, and local blood circulation was improved remarkably. In 3-24 months after transplantation, about 85.2% of patients had remission of hip pain, functional improvement and elongation of walking distance. At 12-24 months after transplantation, X-ray film, CT and MRI examinations indicated that bone matrix in femoral head had changed. For instance, cartilage surface of femoral head was smooth; inflammation of hip cartilage relieved or disappeared; necrotic area was reduced; new bones were formed; fracture line healed.
Animal experiments and clinical observations demonstrated that there were three steps for super-selective stem cell transplantation of blood-supply artery of femoral head to treat femoral head necrosis. The first phase was secretion of cytokines. At 1-21 days after transplantation, stem cells released various angiogeneicsis cytokines, including vascular endothelial growth factors (VEGF), basic fibroblast growth factor (bFGF), hepatic growth factors (HGF) and interleukins, under hypoxic conditions. All those cytokines could promote improvement of local blood circulation and relieve hip pain. However, the new vessels were not generated at this time. The second phase was angiogenesis. At 3-6 months after transplantation, a new vascular net was generated in ischemic femoral head, and this could improve the clinical symptoms. The third phase was bone tissue repair. At 12-24 months after transplantation, femoral head presented histological changes, and new bone was formed. The clinical symptoms were improved remarkably or treated. Stem cell transplantation seems a promising treatment for patients presenting stage Ⅰ-Ⅲa ischemic femoral head necrosis. If femoral head collapsed remarkably, the effect on deformation was poor. All patients in this study did not have any poor responses. Autologous bone marrow mesenchymal stem cell transplantation is considered as convenient and safe, thus it is a novel method to cure ischemic femoral head necrosis. However, some difficulties, such as the prevention of restenosis of newborn vessels and the degree of repair of necrotic bone tissue, need to be further studied.

REFERENCES

1 Li ZR. The ARCO staging system for osteonecrosis of the femoral head.Zhonghua Waike Zazhi 1996;34(3):186-187
2 Yang SH,Yang C,Xu WH,et al. Avascular Necrosis of the Femoral Head Produced in Rabbits by Freezing. Zhonghua Jiaoxing Waike Zazhi 2001;8(1)：48
3 Yang SH. Osteonecrosis.Beijing: People's Military Surgeon Press 2002:117-124
4 Noel D, Djouad F, Jorgense C. Regenerative medicine through mesenchymal stem cells for bone and cartilage repair.Curr Opin Investig Drugs 2002;3(7):1000-1004
5 Gehling Vou, Erguns, Sehumacher V, et al. In vitro differentiation of endothelial cells from AC 133-positive progenitor cells. Blood 2000;95:3106-3112
6 Quirici N, Soligo D, Ganeva L. et al. Differentiation and expansion of endothelial cells from human bone marrow CD 133(+) cells. Br J Heamatol 2001;115:186-194
7 Ikenaga S, Hamano K, Nishida M, et al. Autologous bone marrow implantation induced angiogenesis and improved deteriorated exercise capacity in a rat ischemic hindlimb model.J Surg Res 2001;96(2):277-283
8 Yang XF,Wu YL,Wang HM,et al. Autologous peripheral blood stem cells transplantation in treatment of 62 cases of lower extremity ischemic disorder .Zhonghua Neike Zazhi 2005;44(2):95-98
9 Ji WF,Tong PJ,Zheng WB,et al. Experimental Study on Treatment of Femoral Head Necrosis with Arterial Perfusion of Marrow Stem Cells.Zhongguo Zhongxiyi Jiehe Zazhi 2004;24(11):999-1002

干细胞移植改善股骨头坏死缺血状态的
实验及临床效应：20只模型兔及188例
患者资料分析

杨晓凤，王红梅，许忆峰，张轶斌，吴雁翔，吕 欣，吕乃武，单 鸿，马淑彦
解放军第四六三医院细胞治疗中心，辽宁省沈阳市 110042
杨晓凤,女，1950年生，辽宁省沈阳市人，汉族，1973年白求恩医科大学毕业，主任医师，硕士生导师，主要从事干细胞移植再生医学的研究。
摘要
背景：缺血性股骨头坏死由于股骨头局部滋养血管损伤、供血不足致病，迄今尚无最佳治疗方案，改善股骨头血液供应成为治疗本病的关键。

目的：通过动物实验和临床观察，验证自体骨髓间充质干细胞移植血管再生技术改善股骨头坏死缺血状态的效果。
设计：对比动物实验和自身对照临床观察。
单位：解放军第四六三医院细胞治疗中心。
资料：①动物：选用日本大耳白兔20只,雌雄不拘，体质量3.0~4.0 kg，购于沈阳军区总医院动物实验中心。此动物实验符合动物伦理学要求。②对象：选择2004-07/2007-07在解放军第四六三医院细胞治疗中心住院的具有完整随访资料的缺血性股骨头坏死患者188例（335髋），男113例，女75 例，年龄18~72岁。均经髋关节X线摄片、核素扫描、MRI、CT检查确诊；对治疗和实验知情同意。实验经医院伦理委员会批准。
方法：①动物实验：实验于2004-01/2004-06在沈阳军区总医院动物实验中心完成。采用液氮冷冻股骨头缺血性坏死模型建立方法，双后肢股骨头均制作缺血模型，右侧为移植组，左侧为对照组。抽取骨髓，将所获单个核细胞经右股动脉注入；同法将生理盐水注入左侧股动脉内。②临床观察：密度梯度离心缺血性股骨头坏死患者自体骨髓，分离单个核细胞。行旋股内、旋股外及闭孔动脉干细胞移植术。
主要观察指标：①4周后行DSA动脉造影观察双侧血管新生情况。4，12周后取双侧股骨头标本，行病理切片观察新骨再生及股骨头修复程度。②于移植后第3，6，12，24个月观察患者髋关节疼痛、行走间距及步态、髋关节外展与内旋功能变化。于干细胞移植后6个月行股骨头供血动脉造影术，观察血管新生及股骨头供血动脉充盈情况。于干细胞移植后6，12，24个月行CT，X射线，MRI检查，观察股骨头区形态学及缺血状态变化。于干细胞移植前，移植后3，6，12，24个月进行髋关节Harris评分评价髋关节功能。
结果：动物实验结果：①20只兔均进入结果分析。②DSA动脉造影结果：移植后4周移植组兔右后肢股骨头区供血动脉较对照组股动脉明显增多。③病理结果：移植后12周右侧股骨头软骨、板层骨及骨小梁明显修复，左侧股骨头坏死无改善。临床观察：188例患者均进入结果分析。①症状改善：188例患者中髋关节疼痛缓解164例（87.3%），关节功能改善147例（78.4%），行走间距延长150例（80.0%）。②影像学改变：移植后6个月12例患者DSA动脉造影显示，股骨头供血动脉较移植前明显增多、增粗，血流速度增快；12~24个月后X射线平片，CT，MRI检查24例患者坏死的股骨头区可见骨质病变获得改善。③髋关节Harris评分：干细胞移植后6,12,24个月高于移植前，差异有非常显著性意义(t=-3.423,-6.714,-9.039,P﹤0.01）。
结论：经动脉自体骨髓间充质干细胞移植可改善股骨头坏死缺血状态，是治疗缺血性股骨头坏死的有效手段。
关键词：骨髓间充质干细胞；动脉内干细胞移植；股骨头坏死；血管新生
中图分类号: R394.2 文献标识码: A 文章编号: 1673-8225(2008)08-01558-05
杨晓凤，王红梅，许忆峰，张轶斌，吴雁翔，吕欣，吕乃武，单鸿，马淑彦.干细胞移植改善股骨头坏死缺血状态的实验及临床效应：20只模型兔及188例患者资料分析[J].中国组织工程研究与临床康复，2008，12(8):1558-1562
[www.zglckf.com/zglckf/ejournal/upfiles/08-8/8k-1558(ps).pdf]
(Edited by ANNA D. PETROPOULOU/Ji H/Wang L)