心脏瓣膜组织工程：用人间充质祖细胞对去细胞猪主动脉瓣膜基质进行再接种。

Tissue engineering of cardiac valves: re-seeding of acellular porcine aortic valve matrices with human mesenchymal progenitor cells.

作者信息

Knight Richard L, Booth Catherine, Wilcox Helen E, Fisher John, Ingham Eileen

机构信息

Institute of Medical and Biological Engineering, University of Leeds, Leeds, UK.

出版信息

J Heart Valve Dis. 2005 Nov;14(6):806-13.

PMID:16359063

Abstract

BACKGROUND AND AIM OF THE STUDY

Tissue-engineered heart valves have the potential to overcome the limitations of present heart valve replacements. This study investigated the potential for re-seeding an acellular porcine heart valve matrix using human mesenchymal progenitor cells (MPC).

METHODS

MPC were isolated from the bone marrow of patients undergoing hip replacement operations. Putative MPC were then cultured in several differentiation media in order to determine the multipotential differentiation capacity of the cells. The MPC were also characterized by FACS analysis. Cells at passage 8 were then seeded at between 1 x 10(4) and 1 x 10(5) cells/cm2 onto a decellularized porcine aortic valve matrix, and recellularization of the matrix was assessed. The phenotype of the re-seeded cells and re-seeded cell density was then determined by histology and immunohistochemistry.

RESULTS

Putative MPC were successfully isolated and differentiated into cells of the adipogenic, neurogenic, and myogenic lineages. FACS analysis showed the cells to have a similar phenotype to those isolated by others (CD45-, CD13+, D7FIB+, CD105+, CD10+/-, LNGFR+/-, CD55+, BMP- and AP+/-). Cells seeded onto an acellular valve matrix penetrated the center of the tissue after four weeks to 2% of homograft cell density. Phenotypic analysis of the cells in the re-seeded matrix revealed the cells to have a similar phenotype to native valve interstitial cells (vimentin+, alpha-smooth muscle actin+, heavy chain myosin slow-, desmin-). However, re-seeded cells also expressed osteogenic markers (alkaline phosphatase, osteonectin, and osteopontin).

CONCLUSION

This study has shown, for the first time, that human MPC have the capacity to infiltrate an acellular porcine valve matrix under static conditions in vitro. Future studies will comprise culture under pulsatile flow in a physiological heart valve bioreactor to maintain the desired cell phenotype and increase cell density.

摘要

研究背景与目的

组织工程心脏瓣膜有潜力克服现有心脏瓣膜置换术的局限性。本研究调查了使用人间充质祖细胞（MPC）重新接种去细胞猪心脏瓣膜基质的可能性。

方法

从接受髋关节置换手术患者的骨髓中分离出MPC。然后将假定的MPC在几种分化培养基中培养，以确定细胞的多能分化能力。还用流式细胞术分析对MPC进行了表征。然后将第8代细胞以1×10⁴至1×10⁵个细胞/cm²的密度接种到去细胞猪主动脉瓣膜基质上，并评估基质的再细胞化情况。然后通过组织学和免疫组织化学确定重新接种细胞的表型和重新接种的细胞密度。

结果

成功分离出假定的MPC，并将其分化为脂肪生成、神经生成和成肌谱系的细胞。流式细胞术分析显示这些细胞具有与其他人分离出的细胞相似的表型（CD45⁻、CD13⁺、D7FIB⁺、CD105⁺、CD10⁺/⁻、LNGFR⁺/⁻、CD55⁺、BMP⁻和AP⁺/⁻）。接种到去细胞瓣膜基质上的细胞在4周后渗透到组织中心，达到同种异体移植细胞密度的2%。对重新接种基质中的细胞进行表型分析发现，这些细胞具有与天然瓣膜间质细胞相似的表型（波形蛋白⁺、α平滑肌肌动蛋白⁺、重链肌球蛋白慢⁻、结蛋白⁻）。然而，重新接种的细胞也表达成骨标志物（碱性磷酸酶、骨连接蛋白和骨桥蛋白）。