Experimental Study On The Effect Of Carboxymethyl Chitosan Scaffold Loaded With Platelet-rich Plasma On Diabetic Rats Wound Healing

A refractory wound refers to a type of wound that cannot be healed during physiological healing,and the total healing process is closely related to the individual’s state.As the population ages and the incidence of chronic diseases increases,chronic refractory wounds caused by diseases such as diabetes,malignant tumors and immune function are also showing high incidence.The difficulty in the treatment of refractory wounds lies in the lack of a good blood supply to the wound surface.The skin tissue around the defect is repeatedly stimulated by inflammation,resulting in the formation of scar tissue and shrinking and thinning of the skin and subcutaneous tissue.The main treatment is etiological treatment,local treatment includes removal of necrotic tissue,enhanced dressing,closed negative pressure drainage and hyperbaric oxygen therapy.The traditional dressing method is not ideal because the gauze is easy to colonize bacteria and the new tissue grows into the gauze mesh and the dressing tears cause secondary damage.Therefore,this study aims to develop a kind of bacteriostatic effect that can promote wound healing and bacteriostatic The effect of the absorbable stent for the treatment of refractory wounds.Platelet rich plasma(PRP)is a concentrated suspension of plasma containing high platelet concentrations.After activation of platelets in PRP,various growth factors,tissue factors and antimicrobial peptides that promote wound healing are released,which accelerates the proliferation and differentiation of local repair cells,the synthesis of extracellular matrix and the formation of new blood vessels,thereby enhancing tissue regeneration and repair.ability.In the early stage of this research group,the application of fresh PRP to diabetic foot wounds has achieved good results.However,the fresh PRP was only stored for 5 days under the condition of shaking at 22 ℃,and if it was lyophilized,it was possible to store at room temperature for a long time.The research group conducted PRP freeze-drying research in the early stage,the platelet recovery rate was high,and the concentration of growth factors in platelets remained stable after rehydration.Therefore,freeze-dried platelets can be applied to refractory wound treatment.In view of the long-term nature of refractory wound repair and the burst release of active protein by PRP,prolonging the release time of active protein and maintaining the concentration of active protein during treatment become a further problem to be solved.The application of new biomaterial-loaded active substances in the treatment of damaged wounds has become a research hotspot.Carboxymethyl chitosan(CMCS)is soluble in water,and the dissolved solution is weakly alkaline;its biocompatibility,non-toxicity,moisture retention,bacteriostasis,biodegradability and other excellent properties,The CMCS has been widely used in the fields of medicine,food,and bioengineering.The cross-linking agent of CMCS forms a hydrophobic pore to form a three-dimensional structure,which can be loaded with drugs or biologically active substances,so that the supported scaffold has a certain sustained release property and corresponding mechanical properties,and is suitable for tissue repair.This study intends to load PRP on a CMCS scaffold to make a PRP/CMCS scaffold.The stent slowly releases active protein during the healing process of chronic refractory wounds,thereby continuously promoting wound healing;and PRP in the scaffold can release bacteriostatic peptide,which combines the bacteriostasis of CMCS itself to further enhance the antibacterial effect of the scaffold;After lyophilization,the stent can be stored at room temperature and is easy to carry,further expanding the scope of application.In this study,a new type of scaffold was prepared by exploring the preparation conditions of the new scaffold,and the application effect of the refractory wound was evaluated.A new biomaterial scaffold was explored to be applied to the treatment of chronic refractory wounds.The specific research contents are as follows:Ⅰ.Preparation of CMCS scaffold and screening of optimal ratioDifferent concentrations of CMCS(1%,2%,3%,4%,5%)were cross-linked by different concentrations of Genipin(GNP)(3%,5%,7%,10%)to make different concentrations.The proportioned solution was lyophilized using a freeze dryer to form a CMCS scaffold.According to different pore size and porosity results,3%CMCS and 3%GNP cross-linked scaffold,3%CMCS and 7%GNP cross-linked scaffold,4%CMCS and 5%GNP cross-linked scaffold,4%CMCS and 10%The GNP cross-linked scaffold pore size is between 20 and 125 μ m suitable for skin cell growth.Moreover,the porosity of the above four groups of stents is above 80%,which is in accordance with the pore size of suitable cell growth.Combined with the tensile strength test results,the stent group obtained by cross-linking 4%carboxymethyl chitosan with 5%genipin had the strongest tensile coefficient.In summary,the scaffold obtained by cross-linking 4%carboxymethyl chitosan with 5%genipin was used as the CMCS concentration of the best CMCS scaffold and the cross-linking concentration of genipin for the following experiments.Ⅱ.screening the optimal concentration of PRP to prepare PRP/CMCS stentThe maximum solution volume supported on the CMCS scaffold was obtained based on the swelling rate of the optimal CMCS scaffold.Different concentrations of platelet-rich plasma solution(600 × 109/L,800 × 109/L,1000 × 109/L,1200 × 109/L,1400 × 109/L,1600 × 109/L)were loaded on the above sieved Six groups of carboxymethyl chitosan scaffolds(PRP/CMCS)containing different concentrations of platelet-rich plasma were obtained on the best CMCS scaffold.The above six groups of scaffolds were immersed in the cell culture medium for 24 hours,and the extract was extracted for the culture of 3T3 cells,and the toxic effect and proliferation effect on the cells were detected by CCK-8 assay.Comparing the results with the results of cultured 3T3 cells in cell culture medium,it was concluded that when the concentration of PRP was 1200 × 109/L,the PRP-loaded CMCS scaffold had the best proliferative effect and determined the ratio of PRP/CMCS.The PRP/CMCS scaffold was prepared according to the optimal ratio.Ⅲ.PRP/CMCS stent sustained release performance testThe PDGF and VEGF indicators in the PRP/CMCS scaffold were selected for the sustained release performance test.After immersing the PRP/CMCS scaffold in the cell culture medium for different time periods,the extract was extracted for ELISA detection of PDGF and VEGF growth factors.According to the ELIS A results,the PRP/CMCS scaffold prepared in this experiment was immersed in the medium for 24 hours,that is,the release rate of the above two growth factors was about 30-40%on the first day,and the daily release amount was gradually decreased over time.The release period of the whole growth factor can be as long as 21 days,which has a significant delayed release effect compared with the burst release of fresh PRP.Ⅳ.PRP/CMCS stent inhibition testThe agar plates were prepared by mixing the Escherichia coli solution with the OD value of 0.1 at a wavelength of 450 nm and the Staphylococcus aureus solution,and the prepared scaffold was placed on the surface of the agar plate for antibacterial activity test to measure the inhibition zone.Size,according to its size,analyze the inhibitory effect of the scaffold on the complex bacteria solution.From the results,it can be seen that the PRP/CMCS scaffold has obvious inhibition zone formation,the inhibition zone size is about 7mm on average;the CMCS stent inhibition zone size is about 5mm on average;the freeze-dried PRP has inhibition zone formation,and its size is about 1mm;negative control paper No inhibition zone formed.It can be seen that the PRP/CMCS scaffold has a certain antibacterial effect on Escherichia coli and golden Staphylococcus.Ⅴ.Therapeutic effect of PRP/CMCS stent in refractory wounds of diabetic ratsThe diabetic rats were treated with refractory wounds,and the CMCS scaffold and PRP/CMCS scaffolds were placed at the wound surface for healing observation,and compared with the blank group without any treatment.At the different time points of wound healing(0 days,3 days,7 days,14 days,21 days),the healing effect of wound defect area was evaluated by Image J software.The healing rate of CMCS stent group was significantly increased compared with the blank control group.There was a statistically difference(P<0.001).The healing rate of the PRP/CMCS stent group was significantly higher than that of the CMCS stent group,and there was a statistically difference(P<0.001).Further evaluation from pathological histology:According to HE staining,Masson staining results showed that the inflammatory cells in the PRP/CMCS stent group were significantly reduced,collagen deposition was accelerated and earlier matured,the amount of angiogenesis was increased,and the epidermal formation was complete and uniform.Finally,according to immunohistochemistry and western blot results,the expression of a-SMA,CD31 and TGF-β 3 in the PRP/CMCS stent group was higher than that in the control group,and the expression of IL-6 was lower than that in the control group;a-The protein expression levels of SMA and VEGFA were higher than those of the control group.Therefore,the PRP/CMCS stent group has a certain healing-promoting ability for refractory wounds.In summary,according to the results of pore size,porosity and tensile strength,the best CMCS scaffold ratio was 4%carboxymethyl chitosan crosslinked by 5%genipin.The results of CCK-8 assay showed that the optimal platelet-rich plasma loading concentration was 1200 × 109/L.The sustained release results showed that the PRP/CMCS scaffold can release growth factors for up to 21 days and has a good sustained release effect.The bacteriostatic test proved that the PRP/CMCS scaffold has a certain inhibitory effect on the growth of Escherichia coli and Staphylococcus aureus.The results of the diabetic refractory wound animal experiment showed that the PRP/CMCS scaffold has the effects of inhibiting inflammation,promoting collagen fibril deposition,promoting neovascularization,and promoting the expression of pro-healing protein compared with the CMCS scaffold and the blank group.Therefore,the PRP/CMCS stent has a good effect on promoting wound healing in chronic refractory wounds.This creates new ideas for the treatment of subsequent refractory wounds and provides a basis for clinical applications.