Effect of Relaxin Expression from an Alginate Gel-Encapsulated Adenovirus on Scar Remodeling in a Pig Model

PURPOSE: Relaxin (RLX) is a transforming growth factor-β1 (TGF-β1) antagonist that is believed to function as a potent collagen re-arranger and a major suppressor of extracellular matrix components. Adenoviruses (Ads) are accepted vectors for cancer gene therapy. However, repeated treatments of Ad are limited by short-term biological activity in vivo. The efficacy of sustained RLX expression to scar remodeling was assessed using an injectable alginate gel-matrix system. MATERIALS AND METHODS: Pig scar tissue was treated with relaxin-expressing Ad loaded in alginate gel (gel/Ad-RLX). Surface areas, color, and pliability of scars were compared, and various factors influencing scar formation and collagen arrangement were analyzed. RESULTS: Gel/Ad-RLX decreased scar size, color index, and pliability. Immunohistochemistry showed decreased levels of major extracellular matrix proteins in the gel/Ad-RLX-treated group. Furthermore, treatment with gel/Ad-RLX reduced expression of tissue inhibitor of metalloproteinase-1 and alpha-smooth muscle actin and markedly increased expression of matrix metalloproteinase-1 in pig scar tissues. Gel/Ad-RLX also significantly downregulated TGF-β1 and upregulated TGF-β3 mRNAs in pig scar tissues. CONCLUSION: These results support a prominent role for RLX in scar remodeling and suggest that gel/Ad-RLX may have therapeutic effects on scar formation.

Regeneration of rat corpora cavernosa tissue by transplantation of CD133+ cells derived from human bone marrow and placement of biodegradable gel sponge sheet / 亚洲男科学杂志(英文版)

The objective is to develop an easier technique for regenerating corpora cavernosa tissue through transplantation of human bone marrow-derived CD133+ cells into a rat corpora cavernosa defect model. We excised 2 mm × 2 mm squares of the right corpora cavernosa of twenty-three 8-week-old male nude rats. Alginate gel sponge sheets supplemented with 1 × 104 CD133+ cells were then placed over the excised area of nine rats. Functional and histological evaluations were carried out 8 weeks later. The mean intracavernous pressure/mean arterial pressure ratio for the nine rats (0.34258 ± 0.0831) was significantly higher than that for eight rats with only the excision (0.0580 ± 0.0831, P = 0.0238) and similar to that for five rats for which the penis was exposed, and there was no excision (0.37228 ± 0.1051, P = 0.8266). Immunohistochemical analysis revealed that the nine fully treated rats had venous sinus-like structures and quantitative reverse transcription polymerase chain reaction analysis of extracts from their alginate gel sponge sheets revealed that the amounts of mRNA encoding the nerve growth factor (NGF), and vascular endothelial growth factor (VEGF) were significantly higher than those for rats treated with alginate gel sheets without cell supplementation (NGF: P = 0.0309; VEGF: P < 0.0001). These findings show that transplantation of CD133+ cells accelerates functional and histological recovery in the corpora cavernosa defect model.

The effects of the gel compound from bone marrow mesenchymal stem cells and muscle-like cells/calcium alginate on myoblast formation around urethra in rats of stress urinary incontinence / 中华泌尿外科杂志

ObjectiveTo explore the effects of myoblast formation around the urethra of stress urinary incontinence (SUI) rats after treated with bone marrow mesenchymal stem cells(BMSCs) or musclelike cells/calcium alginate composite gel injection therapy.MethodsIsolation,cultivation and identification of Sprague-Dawley rat bone marrow mesenchymal stem cell were performed.5-azacytidine was introduced to induce muscle-like cells.Calcium alginate gel was initially prepared by 2％ sodium alginate and 1％ calcium chloride solution at a volume ratio of 5∶1.Compounds of stem cells or muscle-like cells were mixed with gel,respectively,and were prepared for microinjection.SUI was produced in 72 6-week-old female Sprague-Dawley rats.The rats were then divided into 4 groups:Gel group,stem cell-gel group,muscle-like cell-gel group and mock control group.Each group was further divided into 3 groups.Submucosal injection of gel was performed at urethra and bladder neck.After preparation of cross sections of rat urinary tract at 4 weeks and 8 weeks after injection,HE staining,fluorescent tracing,staining of Desmin and α-skeletal muscle actin (α-SMA) were performed.OD values of positive rates were compared.ResultsAt 4 weeks and 8 weeks after injection in stem cell-gel group and muscle-like cell-gel group,growth of blood vessels gradually increased at gel edge,BMSCs and muscle-like cells gathered around the new blood vessels observed by fl(u)orescence tracer,muscle-like cells grew into elongated spindle-like cells.Desmin and α-SMA staining were positive in these groups,and the OD values in the stem cell-gel group and muscle-like cell-gel group was significantly higher than that from the gel only group and control group,but no difference was found between stem cell-gel group and muscle-like cell-gel group.ConclusionsCompound of BMSCs,muscle-like cells and calcium alginate composite gel has the potential to differentiate into muscle cells in the microenvironment of SUI rat model.In short term,the myoblast formation potential is the same whether the BMSCs was introduced into the micro-environment in vivo directly,or the BMSCs was implanted into microenvironment after the formation of the muscles cells induced by 5-azacytidine in vitro.

The Effect of Poly-L-lysine on Proliferation and Differentiation of Preadipocyte in the Alginate Gels

Alginate is widely used for scaffold in tissue engineering. However, it has a limitation of cell proliferation due to the lack of cell-to-matrix adhesion. Authors were trying to find out that the alginate gel become an efficient three-dimensional biomatrix in case of mixing with poly-L-lysine (PLL). After harvesting preadipocyte from rat epididymal fat, the proper concentration of PLL for an efficient cell culture was examined in the alginate gel and the level of proliferation of cells were measured in order to find out the efficacy of PLL for the experimental group(alginate/PLL mixed gel) compared to the control group(alginate gel without PLL). In addition, the number of surviving cell was counted and the fat cell stained with oil-red O was observed on the 21st day of the culture. The preadipocytes in the alginate gel were most viable in the PLL concentration of 50 microgram/ml. After 4 days in culture, the level of cell proliferation and the number of preadipocytes were significantly higher in the experimental group than those in the control group. A small number of fat cells stained with oil-red O were starting to be appeared on the 14th day and the larger number of cells on the 21st day of the culture in two groups. These results suggest that PLL increased the proliferation of preadipocyte in the alginate gel through the enhancement of cell-to-matrix adhesion. It also shows that alginate has the advantage of inducing the differentiation of preadipocyte in case of alginate/PLL mixed gel. In conclusion, alginate/PLL mixed gel is turned out to be effective for making a three-dimensional biomatrix.

Growth and Differentiation of Preadipocytes in Alginate and Collagen Gels

Diverse developments in the field of tissue engineering have stimulated much research on tissue production. However, studies on fat tissue still remain insufficient. The purpose of this study is to examine if alginate gel and collagen gel can be used as a three-dimensional scaffold for the culture of preadipocytes, and if these gels can induce preadipocytes to differentiate into mature adipocytes. The preadipocytes harvested from rat epididymal fat pads were three-dimensionally cultured in 1%, 2% alginate gel and collagen gel for 14 days. The morphology, number, and activity of preadipocytes were examined during the experimental period. The results were as follows; 1. The preadipocytes of monolayer culture were spindle shape with rich cytoplasm. The preadipocytes of collagen gel were multipolar or star-like in shape and there was no oil-red 0 stained cell until 14 days. However, the preadipocytes in alginate gel were round and some of cells transformed into mature fat cells which were stained by oil-red 0 after 14 days. 2. The number of preadipocytes in collagen gel continuously increased for 14 days, and significantly increased compared to that of preadipocytes in monolayer culture after 7 days. However, the number of preadipocytes in alginate gel significantly decreased compared to that of preadipocytes in monolayer culture and collagen gel for 14 days, and there was no difference between 1% and 2% alginate gel groups in the number of preadipocytes. 3. The activity of preadipocytes in collagen gel was decreased until 7 days, but not significantly different after 10 days, when compared with that of preadipocytes in monolayer culture. And the activity of preadipocytes in alginate gel was decreased than that of preadipocytes in monolayer culture until 10 days, was higher than that of preadipocytes in collagen gel until 7 days, but was not significantly different compared with that of preadipocytes in monolayer culture and collagen gel on the 14th day. There was no difference between 1% and 2% alginate gel groups in activity of preadipocytes for 14 days. The results suggest that collagen gel are adequate three-dimensional scaffolds in which the proliferation of preadipocytes can be induced, and that alginate gel can be used as a three-dimensional scaffold that has the ability to induce differentiation of preadipocyte although the proliferation of preadipocytes is inhibited.