Adipose tissue-derived stem cells- hydrogel scaffold injection for prevention of esophageal stricture after endoscopic submucosal dissection in a porcine model

Abstract

Esophageal stricture after extensive esophageal endoscopic submucosal dissection (ESD) deteriorates the quality of life, prevention of postoperative strictures has recently been an issue of interest. We hypothesized that adipose tissue-derived stem cells (ADSCs) -hydrogel scaffold mixture can prevent the esophageal stricture. In our study, a biocompatible, injectable and in situ self-crosslinkable hydrogel was designed from a highly auto-oxidative chemical moiety inspired by marine ascidian. First, in vitro test was performed for checking material characterization, biocompatibility and paracrine effects of the hydrogel. The ADSCs encapsulated in the in vivo-crosslinked hydrogel which has similar or slightly higher mechanical property compared to the hydrogel formed by in vivo-mimetic crosslinking would exhibit better paracrine effects. For the next step, in vivo test was performed in mouse model and the hydrogel was successfully crosslinked and stably adhered to the surface of subcutaneous tissues which was confirmed when the injected region was surgically opened after 1-day post-injection. Histological analysis also showed that the hydrogel was tightly integrated onto the tissue and we could conclude that the hydrogel and its crosslinking processes were highly biocompatible even in an in vivo environment. Finally, porcine experiment was performed to determine whether it is effective in an environment similar to the actual clinical situation. Fifteen female domestic pigs weighing 20-25kg were used and they were randomly assigned to a control group (n = 5), ADSCs group (n= 5) or ADSCs-gel group (n=5). On 7, 14, and 21 days after the procedure, the degree of stricture was evaluated by endoscopic and fluoroscopic examination. After evaluation, 2 pigs on day 7, 1 pig on day 14 and 2 pigs on day 21 were sacrificed and histologic evaluation was performed. The mean (± SD) rate of mucosal contraction was 79.5% ±2.0%, 62.8% ± 1.7%, and 37.9% ±2.9% in control, ADSCs group, and ADSCs-gel group, respectively (P < 0.05). Contractions are significantly milder in ADSCs group, and ADSCs-gel group, compared to control (P < 0.05 and P< 0.01) and milder in ADSCs-gel group, compared to ADSCs group (P < 0.05). This effect can imply that ADSCs could be effective to reduce esophageal stricture and this effect can be augmented in ADSCs-Gel mixture. In conclusion, our study demonstrated that ADSCs mixture with ascidianinspired, biocompatible, injectable and in situ self-crosslinkable hydrogel was significantly reduced the post-ESD esophageal stricture by augmenting the paracrine effects of ADSCs and thus may suggest the possibility of using ADSCs-gel mixture to prevent or reduce the esophageal stricture after ESD. 배경: 광범위 식도 내시경점막하박리술 후 발생하는 식도 협착은 삶의 질을 악화시키기 때문에, 협착의 예방은 매우 중요하며, 중간엽줄기세포 주사 또는 경구 복용 등의 방법이 제시되었으나 짧은 지속시간 또는 반복적인 섭취 등의 제한점이 있다. 목적: 본 연구는 지방 조직 유래 줄기 세포-하이드로겔 혼합물의 식도협착예방효과를 확인하고자 하는 대동물 실험으로서, 이를 위하여 in vitro 및 마우스실험 등의 예비실험을 함께 진행하였다. 방법: 멍게의 혈액성분 가운데 강력한 접착성을 가지고 있는 성분을 모사한 하이드로겔의 합성을 1H-NMR과 UV-vis light spectrophotometer 를 통하여 확인하고, in vitro 및 마우스실험을 통하여 줄기세포-하이드로겔의 생체적합성, 파라크린효과 및 조직부착성 등을 확인하였다. 이후 15마리의 돼지 (대조군 5, 줄기세포군 5, 줄기세포-하이드로겔 5마리)에서 원주의 75% 및 길이 3cm의 내시경점막하박리술을 시행하고, 줄기세포 또는 줄기세포-하이드로겔 혼합용액을 절제된 식도의 점막하층에 주입하고 매주 내시경, 투시촬영을 통한 식도협착의 평가를 시행하였다. 1주, 2주, 및 3주차에 각 군에서 2마리, 1마리 및 2마리를 희생하여, 육안적 협착정도와 조직학소견을 비교하였다. 결과: 점막협착비율은 대조군, 줄기세포군 및 줄기세포-하이드로겔군에서 각각 79.5 % ± 2.0 %, 62.8 % ± 1.7 % 및 37.9 % ± 2.9 %였다 (P <0.05). 협착율은 대조군과 비교하였을 때 줄기세포군과 줄기세포-하이드로겔군에서 유의하게 낮았으며, 줄기세포군에 비해 줄기세포-하이드로겔 군에서 유의하게 낮았다. 관찰되는 Q-dot의 수는 줄기세포군과 줄기세포-하이드로겔군 모두 시간이 경과함에 따라 감소하는 양상을 보이지만, 줄기세포군에 비하여 줄기세포-하이드로겔군 에서 관찰되는 Q-dot의 양이 증가되어 있어, 줄기세포-하이드로겔군에서 더 오랫동안, 더 많은 양의 지방유래줄기세포 존재함을 확인하였다. 결론: 본 연구를 통하여 지방유래 줄기세포의 주입이 내시경점막하박리술 후 발생하는 식도 협착을 감소시키는 데 효과적이었고, 줄기세포 단독사용보다는 줄기세포-하이드로겔 혼합물을 사용함으로써 이러한 효과를 증대시킴을 확인하였다.