Cell Based Therapy In Meniscus Tissue Engineering

Abstract

半月軟骨是位於膝關節的纖維軟骨，其損傷在臨床上的治療一直是骨科的挑戰。目前臨床上可以利用一步驟的手術方式將自體的關節軟骨細胞植入缺損的區域。然而，因為半月軟骨手術需要較複雜且精密的技術，而利用組織工程在臨床上應用仍非常有限。雖然許多研究指出關節軟骨細胞與半月軟骨細胞相似，但這些細胞會受不同生長因子與物理性刺激影響，因此本研究將探討半月軟骨與關節軟骨在細胞的特性上可能的異同。進一步將整合半月軟骨的細胞特性與組織工程設計應用於自體半月軟骨細胞植入受損區域之一步驟手術，期能建立以細胞為基礎的治療平台。 本研究首先收集了十位進行全膝關節置換手術之病人的關節軟骨及半月軟骨。我們將半月軟骨分為內、中、外三個區域，連同關節軟骨，各自進行原代細胞培養(primary culture)。各區域細胞的特性會藉由免疫螢光染色及即時聚合酶連鎖反應(real-time PCR)來分析細胞的蛋白質及其相關基因表現。實驗結果顯示內側及中間的半月軟骨細胞擁有關節軟骨細胞的特性。然而，外側的半月軟骨細胞卻有較多纖維母細胞的特性。此外，我們也發現初代的半月軟骨細胞比繼代的半月軟骨細胞有較強的關節軟骨細胞特性。 為了建立能增進半月軟骨損傷癒合的治療方式，我們進行以下動物的實驗設計：將年輕牛隻後腳的半月軟骨細胞植入有安全核可、能於臨床上使用的纖維蛋白膠(fibrin gel)中，並將其植入徑向損傷的半月軟骨( radial meniscus tear)中進行體外培養。實驗分成四組：只植入纖維蛋白膠組、繼代培養兩週的半月軟骨細胞組、過夜分離處理組以及快速分離處理組。首先，我們先藉由半月軟骨細胞的存活率、細胞外基質的結構及基因的表現來決定膠原蛋白酶的使用濃度及使用時間，實驗結果顯示使用0.2%膠原蛋白酶處理30分鐘組（快速分離

組）能有較高的細胞代謝能力。我們藉由組織分析及衍生的黏合指數評估損傷的半月軟骨的癒合能力。在治療處理一週後，不論是快速分離組或是過夜分離組的第一型膠原蛋白和第二型膠原蛋白的表現都明顯地較繼代培養兩週的組別高。在治療四週及八週後，只有快速分離組仍保有較高的第二型膠原蛋白表現。組織化學分析結果顯示在治療一週、四週及八週後，有植入細胞三組的癒合能力都較只植入纖維蛋白膠組好，惟在植入細胞的三組實驗結果之間沒有顯著差異。綜合以上結果，本研究認為快速分離的半月軟骨細胞將可運用於一步驟的半月軟骨損傷修復手術，以增進半月軟骨癒合的能力。 本研究釐清半月軟骨不同區域的細胞基礎特性，更進一步了解半月軟骨與關節軟骨在組織結構上的異同。此外，本研究亦提供半月軟骨損傷一種有潛力應用於臨床上的治療方式，將半月軟骨細胞結合可注射的纖維蛋白膠整合運用在利用關節鏡的臨床手術上。然而，未來在臨床的半月軟骨再生運用上，仍需進一步的探討活體測試與找到最合適的治療方式。

Meniscus tissue engineering has yet to achieve clinical application because it requires sophisticate techniques. Unlike articular cartilage tissue engineering which currently employs autologous chondrocytes implant into the defect in one-stage surgery. Meniscus is a fibrocartilagenous tissue in the knee joint, current knowledge reveals both similarities and differences between meniscus and articular cartilage and this could be a crucial basis for further research and development. Although many studies demonstrated similarity of articular chondrocytes and meniscus cells, those meniscus cells were induced by growth factor or mechanical stimulation for chondrogenic phenotype presentation. We integrated basic knowledge of meniscus biology and tissue engineering to replicate cell based therapy meniscus on single step autologous chondrocyte implantation platform, aiming for utilizing this knowledge from bench work to clinical practice. Fundamental properties of meniscus cells were therefore confirmed. Meniscus and cartilage pieces from 10 subjects during total knee replacements were collected and the cells were isolated, followed by simple expansion. Chondrogenic properties were examined by immunofluorescence and gene expression analyses. The results showed that without chondrogenic induction, inner and middle meniscus cells possess chondrogenic phenotype whereas outer meniscus cells presented towards fibroblast. Specifically, native meniscus cells exhibited more robust chondrogenic potential compared with those passage 2 monolayer cultured. Thereafter, we proceeded to experiment seeded theses cells into the scaffold and applied into meniscus defect to enhance meniscus healing. First of all, optimal concentration and time of treatment by collagenase which is the enzyme using to digest tissue extracellular matrix, were identified by using meniscus cell viability, organization of the extracellular matrix (ECM), and gene expression, as the indicators. Cell metabolism assay, microscopic examination, and quantitative

real-time reverse transcription polymerase chain reaction analysis were also demonstrated. Subsequently, fibrin gel which is commercially available and has safety record in clinical used, was chosen for cells delivery. An explant model of a radial meniscus tear was used to evaluate the effect of a fibrin gel seeded with one the following – (1) fibrin alone, (2) isolated and passaged (P2) meniscus cells, (3) overnight digested tissue, and (4) rapidly dissociated tissue. The quality of in-vitro healing was determined through histological analysis and derivation of an adhesion index. Our study rests showed rapid dissociation in 0.2% collagenase yielded cells with higher levels of metabolism than either 0.1% or 0.5% collagenase. When seeded in a 3-dimensional fibrin hydrogel, both overnight digested and rapidly dissociated cells expressed greater levels of collagens type I and II than P2 meniscal cells at 1 week. At 4 and 8 weeks, collagen type II expression remained elevated only in the rapid dissociation group. Histological examination revealed enhanced healing in all cell-seeded treatment groups over cell-free fibrin controls at weeks 1, 4, and 8, but there were no significant differences across the treatment groups. In conclusion, rapid dissociation of meniscus tissue may provide a single-step approach to augment regenerative healing of meniscus repairs. Our study demonstrated the potential to apply cell based therapy in clinical practice for meniscus lesion. Rapidly dissociated extracellular matrix technique benefited in providing pericellular matrix which promotes chondrogenic properties of meniscus cells and enhances tissue regeneration. Combining these cells with biphasic injectable fibrin hydrogel would be feasible for arthroscopic assist surgery. However, mechanical study and further experiment in in-vivo study are anticipated for the ultimate goal as a clinical employment.