介入主動運動治療結合生醫支架植入應用於骨軟骨缺損之修復效益：迷你豬模型

The effects of active therapeutic exercise together with bio-implant for osteochondral defects repair in mini-pigs

朱致柔(Chih-Jou Chu)

指導教授 ： 張乃仁

高雄醫學大學/醫學院/運動醫學系碩士班/碩士(2019年)

摘要

背景：關節骨軟骨損傷的治療依然是個難題。馬賽克鑲嵌術是臨床上常見的一種治療方式，但存在著兩個修復上的主要問題，一是提供骨軟骨移植物的部位，之後的自癒狀況不好且可能發生軟骨細胞凋亡，二是修復區無法完全填滿的空隙，修復上多生成纖維軟骨而非功能性透明軟骨，且骨軟骨間的整合不好。為解決上述問題，最佳的治療方式還持續在探討。聚乳酸甘醇酸(PLGA)是通過FDA許可的合成生醫材料，而由其製成的PLGA生醫支架已被證實能支持軟骨的生長。運動能提供適當的力學刺激於調節適合軟骨生長的微環境，且具有抗發炎的效果。植入PLGA生醫支架並搭配復健運動於骨軟骨損傷的治療效益已在小動物獲得驗證。若要能運用於臨床，還需經由適合的大動物轉譯模型先行驗證。然而，結合生醫支架及復健運動於治療骨軟骨損傷的相關研究尚未在任何大動物模型被探討。
目的：探討結合PLGA生醫支架與主動復健跑步機運動於迷你豬骨軟骨缺損的修復效益並評估是否可作為馬賽克鑲嵌術的替代療法。
材料與方法：9隻公蘭嶼迷你豬(約8個月大，平均21.3公斤)，隨機分配5隻為運動組(Treadmill, TRE)，4隻為控制組(Sedentary, SED)。為解決馬賽克鑲嵌術提供自體骨軟骨部位其後面臨缺損處病變(donor site morbidity)或軟骨細胞可能凋亡的狀況，本實驗同時在高承重區和低承重區製造骨軟骨缺損。所有豬隻於雙腳膝蓋皆鑽3個直徑6mm深6mm的骨軟骨缺損，位置分別在股骨內髁(Medial condyle of femur, MC)、前內側髕股溝(Medial ridge of patellofemoral groove, MG)及後外側髕股溝(Lateral ridge of patellofemoral groove, LG)，右腳三個缺損會於手術時植入PLGA生醫支架作為PLGA生醫支架組，左腳則為缺損控制組(Empty Defect, ED)。運動組於術後2個月開始進行為期1個月的復健跑步機運動，每天約30分鐘，每週4天，之後籠內自由活動直到術後6個月犧牲。控制組術後一直於籠內自由活動直到術後6個月犧牲。於術後3個月即是復健運動結束時，麻醉所有迷你豬進行平面X光檢查。所有迷你豬於犧牲後進行膝軟骨巨觀分析、超高解析度斷層掃描儀了解新生骨組織、組織學染色觀察細胞型態與膠原蛋白排列、免疫組織化學染色觀察發炎因子及特定蛋白表現，最後對巨觀、組織學和發炎表現做量化評分。
結果：在術後3個月的X光成像中，各組於受損區仍可觀察到清楚的空洞。整體來說，TRE-PLGA組在術後6個月的巨觀與組織學分析中呈現最良好的結果。相對良好的骨軟骨整合、新生軟骨細胞、豐富的糖胺聚醣(glycosaminoglycans, GAGs)與良好的整體膠原蛋白及組織排列，於組織學量化評分也獲得顯著較好的分數。同時，也偵測到膠原蛋白Ⅱ型表現及輕微發炎因子(TNF-α, IL-6)呈現。相反地，在SED組觀察到磨損且有剝離的軟骨表面，修復上主要生成了纖維組織並有發炎細胞聚集，膠原蛋白及組織結構散亂，糖胺聚醣也有流失的狀況。在斷層掃描(micro-CT)下，PLGA組觀察到類軟骨下骨囊腫的表現，最高的骨頭體積比(BV/TV)出現在TRE-ED組。比較位於高承重區MC缺損和位於低承重區MG及LG缺損，整體修復結果上並無明顯統計差異，指出此修復策略適用於高低承重區域。
結論：PLGA生物性支架結合適合的復健跑步機運動能有效修復迷你豬的骨軟骨缺損。然而，如果沒有搭配跑步機運動，單獨使用PLGA生物性支架則無法使骨軟骨順利再生。
臨床意義：本研究驗證了PLGA生物性支架結合復健跑步機運動於骨軟骨損傷之療效，也許未來可作為馬賽克鑲嵌術可行的替代療法。

關鍵字

軟骨 ； 骨軟骨 ； 支架 ； 跑步機運動 ； 膝蓋 ； 大型動物模型

並列摘要

Background: Osteochondral repair remains puzzle. Common clinical treatment of mosaicplasty exists limitations. For instance, after harvesting of osteochondral plugs from the knee joint, the donor site noted bad self-healing which may be associated with chondrocytes apoptosis and the potential remaining empty spaces between osteochondral plugs rely on fibrous repair as well as poor osteochondral integration. The golden alternative therapy is under investigation. The polylactic-co-glycolic acid (PLGA) bio-material has been approved by the US Food and Drug Administration (FDA) and the bioscaffold made by PLGA had proven can support cartilage growth. Exercise provides proper mechanical stimuli to regulate microenvironment for cartilage regeneration and exert anti-inflammatory. The effect of PLGA implant combined with exercise on osteochondral repair had confirmed in small animal model. To further apply on clinical, the large animal translational model is needed to verify. However, the therapeutic effect of bio-implant with treadmill rehabilitation on osteochondral lesions remains uninvestigated in large animal model.
Hypothesis: Supported by PLGA scaffolds with a progressive treadmill rehabilitation may benefit the osteochondral lesions repair and offer a feasible alternative treatment of mosaicplasty.
Study Design: Controlled laboratory study.
Methods: Of 9 mini-pigs were randomly allocated to the treadmill exercise (TRE) group or the sedentary (SED) group. All pigs had three 6 mm × 6 mm osteochondral defects on the bilateral knee of medial condyle (MC), medial ridge of patellofemoral groove (MG) and lateral ridge of patellofemoral groove (LG), respectively. Three defects in the right knee had implanted in the PLGA bioscaffold as the PLGA group, and the left knee had left hollow as the empty defect (ED) group. TRE was performed a 1-month treadmill rehabilitation, about 30 minutes per day, and 4 days per week at 2-months postsurgery. After 3 months postsurgery which was the end of rehabilitation, all mini-pigs were anesthetized for plane X-ray examination. After sacrifice at 6-month postsurgery, macroscopic evaluation, micro CT scanning, histology, and immunohistochemical staining were examined and quantitatively scored.
Results: At 3 months postsurgery, regarding the X-ray imaging, the both PLGA groups and ED groups displayed clear hollow on defect sites. Overall, at 6 months postsurgery, the TRE-PLGA group revealed the favorable results on gross and histological scores corresponding to a sound osteochondral integration, chondrocyte-like cells embedded in lacunae, abundant glycosaminoglycans (GAGs), and collagen alignment. In addition, good collagen type Ⅱ expression and the modest inflammatory response (TNF-α, IL-6) were also detected. In contrast, the SED group revealed an irregular surface with abrasion, fibrotic tissue with some empty void and inflammatory cells as well as unorganized collagen fiber and fewer GAGs. In the micro-CT scanning, the subchondral bone cyst-like pouch was displayed on the PLGA groups, and the highest BV/TV showed on TRE-ED group. Furthermore, compared the high-loaded MC defect to the low-loaded MG and LG defects, there were no statistical differences on overall repair, indicating suitable regenerative strategy for both high weight-bearing and low weight-bearing regions.
Conclusion: PLGA bio-implant combined with proper treadmill rehabilitation can develop a sound result on osteochondral defect repair in mini-porcine. However, without the treadmill rehabilitation, PLGA bio-implant alone generates unfavorable osteochondral regeneration.
Clinical Relevance: The findings demonstrate the therapeutic efficacy and feasibility of PLGA scaffold implantation involving appropriate treadmill rehabilitation for osteochondral regeneration may be an alternative treatment of mosaicplasty.

並列關鍵字

cartilage ； osteochondral ； scaffold ； treadmill exercise ； knee ； large animal model

參考文獻

1. International Cartilage Regeneration & Joint Preservation Society. Available at: https://cartilage.org/patient/about-cartilage/cartilage-repair/mosaicplasty/.

2. Adaes S, Mendonca M, Santos TN, Castro-Lopes JM, Ferreira-Gomes J, Neto FL. Intra-articular injection of collagenase in the knee of rats as an alternative model to study nociception associated with osteoarthritis. Arthritis Res Ther. 2014;16(1):R10.

3. Allen J, Imbert I, Havelin J, et al. Effects of treadmill exercise on advanced osteoarthritis pain in rats. Arthritis Rheumatol. 2017;69(7):1407-1417.

4. Andersen HM-L, J??rgensen E, Dybkjær L, J??rgensen B. The ear skin temperature as an indicator of the thermal comfort of pigs. Applied Animal Behaviour Science. 2008;113(1-3):43-56.

5. Benazzo F, Cadossi M, Cavani F, et al. Cartilage repair with osteochondral autografts in sheep: effect of biophysical stimulation with pulsed electromagnetic fields. J Orthop Res. 2008;26(5):631-642.