Yajuan Song

Identification of the Centrifuged Lipoaspirate Fractions Suitable for Postgrafting Survival.

Background: The Coleman centrifugation procedure generates fractions with different adipocyte and progenitor cell densities. This study aimed to identify all fractions that are feasible for implantation. Methods: Human lipoaspirates were processed by Coleman centrifugation. The centrifugates were divided arbitrarily into upper, middle, and lower layers. Adipocyte viability, morphology, numbers of stromal vascular fraction cells, and adipose-derived mesenchymal stem cells of each layer were determined. The 12-week volume retention of subcutaneously implanted 0.3-ml lipoasperate of each layer was investigated in an athymic mice model. Results: Most damaged adipocytes were located in the upper layers, whereas the intact adipocytes were distributed in the middle and lower layers. A gradient of stromal vascular fraction cell density was formed in the centrifugates. The implant volume retentions of samples from the upper, middle, and lower layers were 33.44 ± 5.9, 55.11 ± 4.4, and 71.2 ± 5.8 percent, respectively. Furthermore, the middle and lower layers contained significantly more adipose-derived stem cells than did the upper layer. Conclusions: The lower layer contains more viable adipocytes and stromal vascular fraction cells leading to the highest implant volume retention, whereas the most impaired cells are distributed in the upper layer, leading to the least volume retention. Although with a lower stromal vascular fraction content, the middle layer has a substantial number of intact adipocytes that are capable of retaining partial adipose tissue volume after implantation, suggesting that the middle layer may be an alternative fat source when large volumes of fat grafts are needed for transplantation.

Intra-Bone Marrow Transplantation of Endosteal Bone Marrow Cells Facilitates Allogeneic Hematopoietic and Stromal Cells Engraftment Dependent on Early Expression of CXCL-12

Background Hematopoietic stem cell transplantation (HSCT) has been considered as an effective approach at inducing allogeneic hematopoietic reconstitution and immune tolerance. However, it remains critical to find the optimal HSCT delivery method and robust sources of hematopoietic stem cells (HSCs). Material/Methods We introduced a new method by infusing allogeneic endosteal bone marrow cells (BMCs) harvested from long bones endosteum through intra-bone marrow transplantation (IBBMT) into irradiated mice. Recipient mice that were transplanted with central BMCs or through intravenous bone marrow transplantation (IVBMT) were used as controls (n=6 per group). We compared the new method with each control group for allogeneic HSCs homing pattern, peripheral blood chimerism level, skin allograft survival time, and donor stromal cell percentage in recipient BM. AMD3100 was injected to determine whether chemokine stromal cell-derived factor-1 (CXCL-12) was critical for the new method. Results More allogeneic HSCs homed into spleen and bone marrow for the new method as compared to each control group. IBBMT of endosteal BMCs led to a higher peripheral blood chimerism and skin allograft survival. At 18 weeks, donor stromal cell percentage in recipient BMCs was higher for the new method than in each control group. By AMD3100 blockade at day 1, peripheral blood chimerism level and donor stromal cell percentage were significantly reduced as compared to the control group without AMD3100 blockade. Conclusions Our study suggests that IBBMT of endosteal BMCs is an effective approach for HSCT in inducing allogeneic hematopoietic reconstitution. The advantage is dependent upon the early expression of CXCL-12 after bone marrow transplantation.

Morphological features of cell death and tissue remolding of fat grafts.

BackgroundFat tissue graft has been commonly used for soft tissue augmentation. However, the mechanisms underlying the maintenance of graft volume and weight are still unclear. As morphological features provide direct evidences for cell death and survival, we aimed to investigate the fate of grafted adipocytes and the dynamic changes in the remodeling of adipose tissues by transmission electron microscopy technique. MethodsThe unilateral inguinal fat pad of C57BL/6J mice was autografted to the dorsa of the mice. Perilipin expression and morphological changes were investigated by immunohistochemistry staining and transmission electron microscopy, respectively, in grafted tissues collected at posttransplantation days 0, 1, 3, 5, 7, 14, and 30. ResultsTransmission electron microscopy analysis revealed that most adipocytes in grafts showed traits of cell death on postgrafting day 3. Multilocular adipocytes with naive nuclei were observed as early as day 5 and a larger number of multilocular adipocytes were found on day 14. Perilipin immunostaining revealed that only some adipocytes located in the margin of grafts survived through the ischemic injury. New adipocytes were visualized at the periphery of the grafts, although the scope of viable adipocyte zonal areas increased from day 5 to day 30. ConclusionsThe results provide ultrastructural evidences associated with the remodeling dynamics of adipose tissue grafts. It is suggested that maximized volume of graft should be obtained through promoting regeneration other than improving survival of grafted adipose tissues.

Local Cytotoxic T-lymphocyte–associated Antigen-4 Immunoglobulin Inhibition of Rejection Response Is Dependent on Indoleamine 2,3-dioxygenase Activities in the Allograft

A previous study showed that local gene transfer of cytotoxic T-lymphocyte-associated antigen-4 immunoglobulin (CTLA4Ig) significantly prolonged the survival time of rat flap allografts. However, the underlying mechanism is not fully understood. Indoleamine 2,3-dioxygenase (IDO) is considered to be able to modulate the unresponsiveness state of allografts. In this study, we tested the expression of the CD80 molecule, IDO mRNA, and the level of the tryptophan metabolite kynurenine with or without the application of the IDO blocker 1-methyl-tryptophan (1-MT) in a rat composite tissue allotransplantation model. CD80 expression could be detected in the allograft. The ration of IDO mRNA/glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA and the level of kynurenine were both enhanced (46.3 ± 8.8 versus 4.6 ± 1.8 and 18.9 ± 1.3 μmol/L versus 2.1 ± 0.2 μmol/L separately) after adenovirus-mediated CTLA4Ig (AdCTLA4Ig) transduction. When 1-MT was applied to the AdCTLA4Ig perfusion recipients, the ration of IDO mRNA/GAPDH mRNA (5.2 ± 2.9) and the level of kynurenine (0.8 ± 0.5 μmol/L) were significantly reduced. Moreover, the allograft survival time was greatly reduced when 1-MT was applied to AdCTLA4Ig perfusion recipients compared to single AdCTLA4Ig perfusion therapy recipients (7.2 days versus 13.6 days). We showed that the inhibitory effect of locally delivered CTLA4Ig is dependent on IDO activities within the allograft.

The Utilization of Perforated Bioinert Chambers to Generate an In Vivo Isolated Space for Tissue Engineering Involving Chondrocytes, Mesenchymal Stem Cells, and Fibroblasts

Vascular invasion and admixture of the nude mouse cells with seeded cells make it difficult to reapply the regenerated tissues to the restoration of host tissue defects. Therefore, a device that is capable of allowing for autologous or allogenic tissue growth while preventing host tissue invasion will be a valuable tool for in vivo tissue engineering. We have previously fabricated a novel silicon-perforated chamber. The aim of this study was to evaluate whether this chamber, after being implanted subcutaneously in experimental animals, would hinder host tissue ingrowth while providing an environment inside its cavity for in vivo growth of either autologous or allogenic implant cells. We found that the chamber did not induce severe foreign body reaction, and the chambers with perforated pores of 1-3 mm in diameter effectively inhibited the host granulation tissue or vascular invasion for as long as 3 months. In addition, the exudates rich in vascular endothelial growth factor, basic fibroblast growth factor, transforming growth factor-β, insulin-like growth factor-1, and platelet-derived growth factor-BB were steadily generated and collected in the chambers. In vitro cell culture studies revealed that the exudates were able to support the viability and proliferation of rabbit chondrocytes, rat mesenchymal stem cells, and human fibroblasts. The results indicate that this novel chamber could potentially provide an environment favorable for in vivo tissue engineering while effectively preventing host tissue or vascular invasion.

Usnic acid inhibits hypertrophic scarring in a rabbit ear model by suppressing scar tissue angiogenesis

Hypertrophic scarring is a common condition in the Chinese population; however, there are currently no satisfactory drugs to treat the disorder. Previous studies showed that angiogenesis plays an important role in the early phase of hypertrophic scarring and inhibition of angiogenesis has been reported as an effective strategy for anti-hypertrophic scar therapy. A recent study showed that usnic acid (UA), an active compound found mainly in lichens, inhibited tumor angiogenesis both in vivo and in vitro. To investigate the therapeutic effects of UA on hypertrophic scarring and to explore the possible mechanism involved, a rabbit ear hypertrophic scar model was established. Scars were treated once a week for four weeks with UA, DMSO or triamcinolone acetonide acetate. Histological evaluation of hematoxylin and eosin staining indicated that UA significantly inhibited hypertrophic scar formation, with obvious reductions in scar height and coloration. The scar elevation index (SEI) was also evidently reduced. Massons trichrome staining showed that UA significantly ameliorated accumulation of collagen tissue. Immunohistochemical analysis of CD31 expression showed that UA significantly inhibited scar angiogenesis. In vitro, UA inhibited endothelial cell migration and tube formation as well as the proliferation of both human umbilical vein endothelial cells and scar fibroblast cells. These results provide the first evidence of the therapeutic effectiveness of UA in hypertrophic scar formation in an animal model via a mechanism that involves suppression of scar angiogenesis.

Inhibition of Lymphatic Drainage With a Self-Designed Surgical Approach Prolongs the Vascularized Skin Allograft Survival in Rats

Abstract Vascularized composite allotransplantation (VCA) is an emerging treatment for significant tissue defects. However, VCAs usually consist of multiple highly antigenic skin tissues. Previous studies have shown that the lymphatic system in skin plays important roles in the initiation of immune responses during acute rejection, by transporting T cells and antigen-presenting dendritic cells to regional lymph nodes. Therefore, we designed a new surgical treatment to inhibit lymphatic drainage of skin allografts and investigated whether this approach could promote the survival of allografts and suppress immunological events after transplantation. This procedure was achieved by connecting the vascularized allografts to recipient tissues with only an annular plastic holder, allowing the minimum of allograft contact with recipients. Our results showed that the self-designed treatment for inhibiting lymphatic drainage promoted the survival of allografts, reduced the serum concentration of IL-2, and decreased the percentage of CD4+CD25+ and CD8+CD25+ from the lymphatic nodes draining the transplantation region. In conclusion, these data suggest that self-designed surgical approach is effective in inhibiting lymphatic drainage of skin allografts, and the lymphatic system may be new therapeutic targets for developing techniques or drugs against acute rejection after VCAs.