| Objective:Concentrated growth factors(CGF)were developed by Sacco in 2006 which can improve soft or hard tissue regeneration and have different characteristic.CGF was thought to be an advanced form of platelet-rich plasma to eliminate xenofactors such as bovine thrombin,and mainly used as a source of growth factors for tissue engineering.However CGF in a compressed membrane-like form cannot be served as a qualified barrier membrane,because it may be degraded within 2 weeks or less at implantation sties.So it is unable to provide enough time and space for bone regeneration.In this study,we developed and optimized a heat-compressed technique and test the bioactivity and biocompatibility of heat-compression CGF,to provide experimental basis of possibility and methods for clinical intervention.Methods:Part1.Heat-compression CGF preparation:After compressed with dry gauze,the CGF membranes were compressed by two heat-regulating iron for leather.The surface temperature was set at 2 levels(90,120℃)which were checked by a thermometer.The CGF membranes were put into the ultraviolet-sterilized plastic wrap that was made of polyvinylidene chloride before heat-compression process and then heated in a period of 2-10s.And then heat-compression CGFs were punched into 10mm disks by a dermal biopsy punch.Part2.Degradation in vitro test:Blood were collected from 6 young healthy volunteers aged 24-26(all male)and then immediately centrifuged in a special process by a Medifuge centrifugation system.The different CGF membranes were put into artificial saliva and stored in 37℃ thermostat water bath.Their appearance was photographed then were dried with sterilized gauze and weighted every 48hours.As for subcutaneous implantation test,twelve New Zealand White rabbits weighing 3.5 to 4 kg were randomly divided into 4 groups of 3 animals each and housed at least 2 week prior to the experiment.Intramuscular ketamine(35 mg/kg)injection was used for general anesthesia and got blood samples from the jugular vein.The preparation of CGF was introduced above.After disinfecting the surgical area of a rabbits,two skin incision was made in the abdomen between the midline,and two types of CGF membrane disks(diameter 10 mm)were implanted into the subcutaneous tissue.Rabbits were euthanized at intervals of 1-4 weeks after implantation.Implanted CGF disks were harvested along with part of the surrounding connective tissue,fixed in 4%paraformaldehyde,dehydrated,embedded in paraffin,and sectioned sagittally at a thickness of 6 mm.Sections were stained with either hematoxylin and eosin(HE)or Masson’s trichrome(MT).Part3.Bioactivity in vitro test:Blood samples were collected from 6 young healthy volunteers aged 24-26(all male).Samples were placed into a shaking incubator at 37℃ to allow for growth factor released into the culture media.At 1,3,7,14 days,the 5 mL of culture media was collected,frozen,and replaced with 5 mL of additional culture media.Protein quantification was carried out using ELISA.At desired time points,PDGF-AB and TGF-β were quantified using an ELISA assays according to manufacturer’s protocol as previously described.All samples were measured in triplicate and three independent experiments were performed for each platelet concentrate.Statistical analysis was performed by two-way ANOVA with Bonferroni test.Part4.Animal test used as a GBR membrane:8 New Zealand white rabbits were housed at least 2 week prior to the experiment.Intramuscular ketamine(35 mg/kg)injection was used for general anesthesia.A sagittal incision was made over the scalp of the animal,and a full thickness flap was reflected.Four 6mm diameter circular bony defects were made symmetrically in both parietal bones using a dental trephine bur.As strict inclusion criteria,the dura was not violated.At 6 weeks after surgery was performed,the animals were euthanized.The skin was dissected,and the defect sites were removed along with surrounding bone using a high-speed dental bur.The biopsied specimens were fixed in 10%formalin and transferred to the laboratory for Micro-CT and histomorphometric analysis.Results:1 Degradation testControl group is different to Group A1,B1,C1,D1.As for the accumulated time of degradation,control group is statistically different to Group A,1 B1,C1,D1 and Group C1 is not statistically different to Group D1.In vivo,the gauze-compressed CGF completely degraded within 1-2 weeks,whereas the heat-compressed CGF could keep integrity at least for 3 weeks.2 Bioactivity in vitro testAll groups released the highest cytokines at first day.Control group released more cytokines than all treatment groups.At 21 day,Group C2 and Group D2 even released more cytokines than control group.As for accumulated PDGF-AB,Group B2,C2,D2 released 52.2%,45.0%,13.9%,10.6%PDGF-AB compared to control group.Group B2,C2,D2 released 62.3%,47.6%,21.3%,21.4%cytokine compared to control group when related to accumulated TGF-β.3 Animal test used as a GBR membraneBlank group:Only fibrous connective tissue was observed between the surgical bone margins.Control group:fibrous connective tissue that invaded between the surgical margins was less than blank group.New bone formation was observed away from the surgical margins.Osteogenous activity was found in the margins and calcified bone particles with no connection to the existing bone tissue were detected.Lyoplant group:Fibrous connective tissue that invaded between the surgical margins was less than control group.New bone formation was observed away from the surgical margins.The voids of bone graft was surrounded by osteoclast cells and can observe thin and unmature woven bone and new blood vessel.Heated-compressed CGF group:Fibrous connective tissue that invaded between the surgical margins was less than control group.New bone formation was observed away from the surgical margins.Lots of woven bone and new blood vessel was observed.Conclusion:1 heated-compressed process can delay the degradation of CGF2 the longer heated time,the longer degradation time of CGF3 Bioactivity decreases after heated-compressed process4 heated-compressed CGF can be a qualified barrier membrane... |
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