Peri-implant keratinized gingiva augmentation using xenogeneic collagen matrix and platelet-rich fibrin:A case report

BACKGROUND Keratinized gingival insufficiency is a disease attributed to long-term tooth loss,can severely jeopardizes the long-term health of implants.A simple and effective augmentation surgery method should be urgently developed.CASE SUMMARY A healthy female patient,45-year-old,requested implant restoration of the her left mandibular first molar and second molar.Before considering a stage II,as suggested from the probing depth measurements,the widths of the mesial,medial,and distal buccal keratinized gingiva of second molar(tooth#37)were measured and found to be 0.5 mm,0.5 mm,and 0 mm,respectively.This suggested that the gingiva was insufficient to resist damage from bacterial and mechanical stimulation.Accordingly,modified apically repositioned flap(ARF)surgery combined with xenogeneic collagen matrix(XCM)and platelet-rich fibrin(PRF)was employed to increase the width of gingival tissue.After 1 mo of healing,the widths of mesial,medial,and distal buccal keratinized gingiva reached 4 mm,4 mm,and 3 mm,respectively,and the thickness of the augmented mucosa was 4.5 mm.Subsequently,through the second-stage operation,the patient obtained an ideal soft tissue shape around the implant.CONCLUSION For cases with keratinized gingiva widths around implants less than 2mm􀀀the soft tissue width and thickness could be increased by modified ARF surgery combined with XCM and PRF.Moreover,this surgery significantly alleviated patients’pain and ameliorated oral functional comfort.

Repairing whole facial nerve defects with xenogeneic acellular nerve grafts in rhesus monkeys

Acellular nerve allografts conducted via chemical extraction have achieved satisfactory results in bridging whole facial nerve defects clinically,both in terms of branching a single trunk and in connecting multiple branches of an extratemporal segment.However,in the clinical treatment of facial nerve defects,allogeneic donors are limited.In this experiment,we exposed the left trunk and multiple branches of the extratemporal segment in six rhesus monkeys and dissected a gap of 25 mm to construct a monkey model of a whole left nerve defect.Six monkeys were randomly assigned to an autograft group or a xenogeneic acellular nerve graft group.In the autograft group,the 25-mm whole facial nerve defect was immediately bridged using an autogenous ipsilateral great auricular nerve,and in the xenogeneic acellular nerve graft group,this was done using a xenogeneic acellular nerve graft with trunk-branches.Examinations of facial symmetry,nerve-muscle electrophysiology,retrograde transport of labeled neuronal tracers,and morphology of the regenerated nerve and target muscle at 8 months postoperatively showed that the faces of the monkey appeared to be symmetrical in the static state and slightly asymmetrical during facial movement,and that they could actively close their eyelids completely.The degree of recovery from facial paralysis reached House-Brackmann grade II in both groups.Compound muscle action potentials were recorded and orbicularis oris muscles responded to electro-stimuli on the surgical side in each monkey.Fluoro Gold-labeled neurons could be detected in the facial nuclei on the injured side.Immunohistochemical staining showed abundant neurofilament-200-positive axons and soluble protein-100-positive Schwann cells in the regenerated nerves.A large number of mid-graft myelinated axons were observed via methylene blue staining and a transmission electron microscope.Taken together,our data indicate that xenogeneic acellular nerve grafts from minipigs are safe and effective for repairing whole facial nerve defects in rhesus monkeys,with an effect similar to that of autologous nerve transplantation.Thus,a xenogeneic acellular nerve graft may be a suitable choice for bridging a whole facial nerve defect if no other method is available.The study was approved by the Laboratory Animal Management Committee and the Ethics Review Committee of the Affiliated Wuxi No.2 People's Hospital of Nanjing Medical University,China(approval No.2018-D-1)on March 15,2018.

Bone Regeneration Enhanced by Antigen-Extracted Xenogeneic Cancellous Bone Graft with rhBMP-2 in Rabbits Mandibular Defect Repair

The effects of large piece xenogeneic bone which was separated from healthy pigs as a scaffold on repair of mandibular defect was investigated and the applicability of antigen-extracted xenogeneic cancellous bone (AXCB) soaked with rhBMP-2 in bone defect repair was assessed. Mandibular defects were created in 48 New Zealand Rabbits, and then randomly divided into 4 groups, which was grafted in the mandibular defects with AXCB, AXCB soaked with rhBMP-2, autograft bone, or blank. Equal number of animals from each group was classified into three time points (4, 8, and 12 weeks) after operation for gross pathological observation, hematoxylin and eosin (H & E) staining, radiographic examination, and bone density measurement. H & E staining revealed that the area percentage of bone regeneration in the group of AXCB/rhBMP-2 graft was 27.72 ± 4.68, 53.90 ± 21.92, and 77.35 ± 9.83 when at 4, 8, and 12 weeks, which was better than that of auto bone graft, prompting that the group of AXCB/rhBMP-2 graft had commendable osteogenic effect. And comparing with the AXCB without rhBMP-2, of which the area percentage of bone regeneration was only 14.03 ± 5.02, 28.49 ± 11.35, and 53.90 ± 21.92, the osteogenic effect of AXCB/rhBMP-2 graft was demonstrated to be much better. In the group of AXCB/rhBMP-2 graft, the area percentage of bone regeneration increased, and the implanted materials were gradually degraded and replaced by autogenous bone regeneration over time. We concluded that antigen-extracted xenogeneic cancellous bone (AXCB) graft soaked with rhBMP-2 had shown excellent osteogenic effect in repair of bone defects, with good biocompability.

Xenogeneic stem cell transplantation:Research progress and clinical prospects

Organ transplantation is the ultimate treatment for end-stage diseases such as heart and liver failure.However,the severe shortage of donor organs has limited the organ transplantation progress.Xenogeneic stem cell transplantation provides a new strategy to solve this problem.Researchers have shown that xenogeneic stem cell transplantation has significant therapeutic effects and broad application prospects in treating liver failure,myocardial infarction,advanced type 1 diabetes mellitus,myelosuppression,and other end-stage diseases by replacing the dysfunctional cells directly or improving the endogenous regenerative milieu.In this review,the sources,problems and solutions,and potential clinical applications of xenogeneic stem cell transplantation will be discussed.

High quality repair of osteochondral defects in rats using the extracellular matrix of antler stem cells

BACKGROUND Cartilage defects are some of the most common causes of arthritis.Cartilage lesions caused by inflammation,trauma or degenerative disease normally result in osteochondral defects.Previous studies have shown that decellularized extracellular matrix(ECM)derived from autologous,allogenic,or xenogeneic mesenchymal stromal cells(MSCs)can effectively restore osteochondral integrity.AIM To determine whether the decellularized ECM of antler reserve mesenchymal cells(RMCs),a xenogeneic material from antler stem cells,is superior to the currently available treatments for osteochondral defects.METHODS We isolated the RMCs from a 60-d-old sika deer antler and cultured them in vitro to 70%confluence;50 mg/mL L-ascorbic acid was then added to the medium to stimulate ECM deposition.Decellularized sheets of adipocyte-derived MSCs(aMSCs)and antlerogenic periosteal cells(another type of antler stem cells)were used as the controls.Three weeks after ascorbic acid stimulation,the ECM sheets were harvested and applied to the osteochondral defects in rat knee joints.RESULTS The defects were successfully repaired by applying the ECM-sheets.The highest quality of repair was achieved in the RMC-ECM group both in vitro(including cell attachment and proliferation),and in vivo(including the simultaneous regeneration of well-vascularized subchondral bone and avascular articular hyaline cartilage integrated with surrounding native tissues).Notably,the antler-stem-cell-derived ECM(xenogeneic)performed better than the aMSC-ECM(allogenic),while the ECM of the active antler stem cells was superior to that of the quiescent antler stem cells.CONCLUSION Decellularized xenogeneic ECM derived from the antler stem cell,particularly the active form(RMC-ECM),can achieve high quality repair/reconstruction of osteochondral defects,suggesting that selection of decellularized ECM for such repair should be focused more on bioactivity rather than kinship.

IL-21 accelerates xenogeneic graft-versus-host disease correlated with increased B-cell proliferation

Graft-versus-host disease(GVHD)is a prevalent and potential complication of hematopoietic stem cell trans-plantation.An animal model,xenogeneic GVHD(X-GVHD),that mimics accurately the clinical presentation of GVHD would provide a tool for investigating the mechanism involved in disease pathogenesis.Murine models indi-cated that inhibiting IL-21 signaling was a good therapy to reduce GVHD by impairing T cell functions.We sought to investigate the effect of exogenous human IL-21 on the process of X-GVHD.In this study,human IL-21 was expressed by hydrodynamic gene delivery in BALB/c-Rag2−/−IL-2Rγc−/−(BRG)immunodeficient mice which were intravenously transplanted human peripheral blood mononuclear cells(hPBMCs).We found that human IL-21 exacerbated X-GVHD and resulted in rapid fatality.As early as 6 days after hPBMCs transplanted to BRG mice,a marked expansion of human CD19+B cells,but not T cells,was observed in spleen of IL-21-treated mice.Com-pared with control group,IL-21 induced robust immuno-globulin secretion,which was accompanied by increased accumulation of CD19+CD38high plasma cells in spleen.In addition,we demonstrated that B-cell depletion was able to ameliorate X-GVHD.These results are the fi rst to fi nd in vivo expansion and differentiation of human B cells in response to IL-21,and reveal a correlation between the expansion of B cells and the exacerbation of xenogeneic GVHD.Our fi ndings show evidence of the involvement of B cells in X-GVHD and may have implications in the treat-ment of the disease.

Recruited CD68^(+)CD206^(+)macrophages orchestrate graft immune tolerance to prompt xenogeneic-dentin matrix-based tooth root regeneration

Successful regenerative medicine strategies of xenogeneic extracellular matrix need a synergistic balance among inflammation,fibrosis,and remodeling process.Adaptive macrophage subsets have been identified to modulate inflammation and orchestrate the repair of neighboring parenchymal tissues.This study fabricated PPARγ-primed CD68+CD206+M2 phenotype(M2γ),and firstly verified their anti-inflammatory and tissue-regenerating roles in xenogeneic bioengineered organ regeneration.Our results showed that Th1-type CD3^(+)CD8^(+)T cell response to xenogeneic-dentin matrix-based bioengineered root complex(xeno-complex)was significantly inhibited by M2γmacrophage in vitro.PPARγactivation also timely recruited CD68^(+)CD206^(+)tissue macrophage polarization to xeno-complex in vivo.These subsets alleviated proinflammatory cytokines(TNF-α,IFN-γ)at the inflammation site and decreased CD3^(+)CD8^(+)T lymphocytes in the periphery system.When translated to an orthotopic nonhuman primate model,PPARγ-primed M2 macrophages immunosuppressed IL-1β,IL-6,TNF-α,MMPs to enable xeno-complex to effectively escape immune-mediated rejection and initiate graft-host synergistic integrity.These collective activities promoted the differentiation of odontoblast-like and periodontal-like cells to guide pulp-dentin and cementum-PDLs-bone regeneration and rescued partially injured odontogenesis such as DSPP and periostin expression.Finally,the regenerated root showed structure-biomechanical and functional equivalency to the native tooth.The timely conversion of M1-to-M2 macrophage mainly orchestrated odontogenesis,fibrogenesis,and osteogenesis,which represents a potential modulator for intact parenchymal-stromal tissue regeneration of targeted organs.

Frozen bean curd-inspired xenogeneic acellular dermal matrix with triple pretreatment approach of freeze–thaw,laser drilling and ADSCs pre-culture for promoting early vascularization and integration

Xenogeneic acellular dermal matrix(ADM)is widely used in clinical practice given its good biocompatibility and biomechanical properties.Yet,its dense structure remains a hindrance.Incorporation of laser drilling and pre-culture with Adipose-derived stem cells(ADSCs)have been attempted to promote early vascularization and integration,but the results were not ideal.Inspired by the manufacturing procedure of frozen bean curd,we proposed a freeze–thaw treatment to enhance the porosity of ADM.We found that the ADM treated with-80℃3Rt-30℃3R had the largest disorder of stratified plane arrangement(deviation angle 28.6%)and the largest porosity(96%),making it an optimal approach.Human umbilical vein endothelial cells on freeze–thaw treated ADM demonstrated increased expression in Tie-2 and CD105 genes,proliferation,and tube formation in vitro compared with those on ADM.Combining freeze–thaw with laser drilling and pre-culture with ADSCs,such tri-treatment improved the gene expression of proangiogenic factors including IGF-1,EGF and vascular endothelial growth factor,promoted tube formation,increased cell infiltration and accelerated vascularization soon after implantation.Overall,freeze–thaw is an effective method for optimizing the internal structure of ADM,and tri-treatments may yield clinical significance by promoting early cell infiltration,vascularization and integration with surrounding tissues.

Human mesenchymal stem cells derived from umbilical cord and bone marrow exert immunomodulatory effects in different mechanisms

BACKGROUND Mesenchymal stem cells(MSCs)are an attractive tool to treat graft-versus-host disease because of their unique immunoregulatory properties.Although human bone marrow-derived MSCs(BM-MSCs)were the most widely used MSCs in cell therapy until recently,MSCs derived from human umbilical cords(UC-MSCs)have gained popularity as cell therapy material for their ethical and noninvasive collection.AIM To investigate the difference in mechanisms of the immunosuppressive effects of UC-MSCs and BM-MSCs.METHODS To analyze soluble factors expressed by MSCs,such as indolamine 2,3-dioxygenase,cyclooxygenase-2,prostaglandin E2 and interleukin(IL)-6,inflammatory environments in vitro were reconstituted with combinations of interferon-gamma(IFN-γ),tumor necrosis factor alpha and IL-1βor with IFN-γalone.Activated T cells were cocultured with MSCs treated with indomethacin and/or anti-IL-10.To assess the ability of MSCs to inhibit T helper 17 cells and induce regulatory T cells,induced T helper 17 cells were cocultured with MSCs treated with indomethacin or anti-IL-10.Xenogeneic graft-versus-host disease was induced in NOG mice(NOD/Shi-scid/IL-2Rγnull)and UC-MSCs or BM-MSCs were treated as cell therapies.RESULTS Our data demonstrated that BM-MSCs and UC-MSCs shared similar phenotypic characteristics and immunomodulation abilities.BM-MSCs expressed more indolamine 2,3-dioxygenase after cytokine stimulation with different combinations of IFN-γ,tumor necrosis factor alpha-αand IL-1βor IFN-γalone.UC-MSCs expressed more prostaglandin E2,IL-6,programmed death-ligand 1 and 2 in the in vitro inflammatory environment.Cyclooxygenase-2 and IL-10 were key factors in the immunomodulatory mechanisms of both MSCs.In addition,UC-MSCs inhibited more T helper 17 cells and induced more regulatory T cells than BM-MSCs.UC-MSCs and BM-MSCs exhibited similar effects on attenuating graft-versus-host disease.CONCLUSION UC-MSCs and BM-MSCs exert similar immunosuppressive effects with different mechanisms involved.These findings suggest that UC-MSCs have distinct immunoregulatory functions and may substitute BM-MBSCs in the field of cell therapy.

Preliminary studies of constructing a tissue-engineered lamellar corneal graft by culturing mesenchymal stem cells onto decellularized corneal matrix

AIM:To construct a competent corneal lamellar substitute in order to alleviate the shortage of human corneal donor.METHODS:Rabbit mesenchymal stem cells(MSCs)were isolated from bone marrow and identified by flow cytometric,osteogenic and adipogenic induction.Xenogenic decellularized corneal matrix(XDCM)was generated from dog corneas.MSCs were seeded and cultured on XDCM to construct the tissueengineered cornea.Post-transplantation biocompatibility of engineered corneal graft were tested by animal experiment.Rabbits were divided into two groups then underwent lamellar keratoplasty(LK)with different corneal grafts:1)XDCM group(n=5):XDCM;2)XDCM-MSCs groups(n=4):tissue-engineered cornea made up with XDCM and MSCs.The ocular surface recovery procedure was observed while corneal transparency,neovascularization and epithelium defection were measured and compared.In vivo on focal exam was performed 3 mo postoperatively.RESULTS:Rabbit MSCs were isolated and identified.Flow cytometry demonstrated isolated cells were CD90 positive and CD34,CD45 negative.Osteogenic and adipogenic induction verified their multipotent abilities.MSC-XDCM grafts were constructed and observed.In vivo transplantation showed the neovascularization in XDCMMSC group was much less than that in XDCM group postoperatively.Post-transplant 3-month confocal test showed less nerve regeneration and bigger cell-absent area in XDCM-MSC group.CONCLUSION:This study present a novel corneal tissue-engineered graft that could reduce post-operatively neovascularization and remain transparency,meanwhile shows that co-transplantation of MSCs may help increase corneal transplantation successful rate and enlarge the source range of corneal substitute to overcome cornea donor shortage.

A comparative study of human and porcine-derived decellularised nerve matrices

Decellularised extracellular matrix(dECM)biomaterials originating from allogeneic and xenogeneic tissues have been broadly studied in the field of regenerative medicine and have already been used in clinical treatments.Allogeneic dECMs are considered more compatible,but they have the drawback of extremely limited human tissue sources.Their availability is also restricted by the health and age of the donors.To investigate the viability of xenogeneic tissues as a substitute for human tissues,we fabricated both porcine decellularised nerve matrix(pDNM)and human decellularised nerve matrix for a comprehensive comparison.Photomicrographs showed that both dECM scaffolds retained the ECM microstructures of native human nerve tissues.Proteomic analysis demonstrated that the protein compositions of both dECMs were also very similar to each other.Their functional ECM contents effectively promoted the proliferation,migration,and maturation of primary human Schwann cells in vitro.However,pDNM contained a few antigens that induced severe host immune responses in humanised mice.Interestingly,after removing theα-galactosidase antigen,the immune responses were highly alleviated and the pre-treated pDNM maintained a human decellularised nerve matrix-like pro-regenerative phenotype.Therefore,we believe that anα-galactosidase-free pDNM may serve as a viable substitute for human decellularised nerve matrix in future clinical applications.

In Vivo Confocal Microscopic Observation of Lamellar Corneal Transplantation in the Rabbit Using Xenogenic Acellular Corneal Scaffolds as a Substitute

Background： The limiting factor to corneal transplantation is the availability of donors. Research has suggested that xenogenic acellular corneal scaffolds （XACS） may be a possible alternative to transplantation. This study aimed to investigate the viability of performing lamellar corneal transplantation （LCT） in rabbits using canine XACS. Methods： Fresh dog corneas were decellularized by serial digestion, and LCT was performed on rabbit eyes using xenogeneic decellularized corneal matrix. Cellular and morphological changes were observed by slit-lamp, light, and scanning electron microscopy at 7, 30 and 90 days postoperatively, lmmunocytochemical staining for specific markers such as keratin 3, vimentin and MUC5AC, was used to identify cells in the graft. Results： Decellularized xenogenic corneal matrix remained transparent for about l-month after LCT. The recipient cells were able to survive and proliferate into the grafts. Three months after transplantation, grafts had merged with host tissue, and graft epithelialization and vascularization had occurred. Corneal nerve fibers were able to grow into the graft in rabbits transplanted with XACS. Conclusions： Xenogenic acellular corneal scaffolds can maintain the transparency of corneal grafts about 1-month and permit growth of cells and nerve fibers, and is, therefore, a potential substitute or carrier for a replacement cornea.

Enhanced critical-sized bone defect repair efficiency by combining deproteinized antler cancellous bone and autologous BMSCs

Previously we have demonstrated that calcinated antler cancellous bone（CACB） has great potential for bone defect repair,due to its highly similar composition and architecture to natural extracellular bone matrix.This study is aiming at seeking for an optimal strategy of combined application of CACB and bone marrow mesenchymal stem cells（BMSCs） in bone defect repair.In vitro study demonstrated that CACB promoted the adhesion,spreading and viability of BMSCs.Increased extracellular matrix production and expression of osteogenic markers in BMSCs were observed when seeded on CACB scaffolds.The cells ceased to proliferation in the dual effect of CACB and osteogenic induction at the early stage of incubation.Hence synergistic effect of CACB combined with autologous undifferentiated BMSCs in rabbit mandible critical-sized defect repair was further evaluated.Histological analysis results showed that loading the CACB with autologous BMSCs resulted in enhanced new bone formation and angiogenesis when compared with implanted CACB alone.These findings indicate that the combination of CACB and autologous BMSCs should become potential routes to improve bone repair efficiency

Biomechanical Properties of Transplanted Xenogenic Meniscal Tissue

The clinical repair approaches to meniscal lesions include total or subtotal meniscectomy, transplantation, and tissue engineering. The investigations found that the transplanted xenogenic meniscal tissues, which were treated by60 Co irradiation and deep freezing, maybe one of the effective approaches. In this paper, we evaluated the biomechanical properties of the transplanted xenogenic meniscal tissues at postoperative 1 year. In vitro tensile and compressive tests are performed to compare the properties of tensile elasticity, tensile strength, and compressive elasticcity,between three groups: RAB group of normal rabbit meniscus tissue, Allo group of transplanted allograft meniscal tissue, and Xeno group of transplanted xenogenic meniscal tissue. The meniscus of the Xeno group showed similar tension and compression modulus as the native rabbit meniscus without significant difference(p>0.05),and the tension strength of both the Xeno group and the Allo group were less than that of the native rabbit meniscus(0.05<p<0.1). No significant difference was found between the Xeno group and the Allo group with regard to each biomechanical parameter(p>0.05). These base studies will be helpful in future transplantation and tissue engineering efforts.