Construction And Efficacy Evaluation Of Engineered Biomimetic Cartilage Scaffold With Inflammation-ameliorating Function

The treatment of articular cartilage(AC)defects is one of the most challenging clinical problems.Due to the avascular,aneural,and alymphatic nature,AC shows a limited capacity for self-repair after injury.Inflammtory response is a key player affecting the process of cartilage damage.A large number of pro-inflammatory factors from the inflammatory response will elicit cellular metabolic disorders and exacerbate decomposition of cartilage matrix,ultimately leading to the failure of cartilage repair.In this sense,how to effectively regulate the inflammatory response is an important issue in cartilage defect repair.Over the past decades,many tissue-engineered scaffolds with the characteristics of biomimicking the natural cartilage extracellular matrix(ECM)were constructed for repairing damaged cartilage.Among them,biodegradable aliphatic polyesters,in particular,polylactide(PLA),polyglycolide(PGA),and their copolymer poly(lactide-co-glycolide)(PLGA),are most often used to fabricate electrospun fibrous scaffolds,which structurally resemble the collagen fibers found in the cartilage ECM.However,these polyesters often give rise to severe aseptic inflammation because of their acidic degradation products(e.g.,lactic acid,glycolic acid and/or their oligomers)resultant acidic microenvironment.Meanwhile,the scaffolds per se upon being implanted tend to cause early inflammation due to the innate immune response in the body.As a result,how to construct biomimetic cartilage scaffolds with inflammation-ameliorating capability is the key to achieving efficacious repair of cartilage defects.To this end,by taking in situ acidity neutralization and introduction of biochemical cues as the main strategies,current dissertation research proposed to construct electrospun biodegradable polyester fibers-based biomimetic cartilage scaffolds with inflammation-ameliorating function for cartilage regeneration.The details are outlined as follows:(1)From the perspective of in situ neutralizing acidic degradation products of the aliphatic polyesters,we proposed the concept of “pH-neutral fibers” and developed unidirectional shell-core structured fibers of chitosan/poly(lactide-co-glycolide)(CTS/PLGA)by coaxial electrospinning method.Acid-neutralizing effect of the chitosan shell on the degradation products of PLGA core from in vitro degradation test,and inflammation-ameliorating effect of such “pH-neutral fibers” at both the cellular and tissue levels were successively performed.Results showed that the CTS-shell could obviously hindered the pH decrease caused by the degradation of PLGA-core.After a period of 8-week of degradation,the pH of CTS/PLGA relevant groups was maintained close to neutral,while the PLGA group dropped to pH 3.1.In a mocked acidic environment by using degradation solutions,chitosan-enabled acidity neutralization effectively reduced the secretion of inflammatory factors(37% for IL-6 and 34% for IL-8),downregulated the expression of inflammatory genes(34% for IL-6,69% for IL-8 and 56% for IL-1β)and upregulated the expression of ECM relevant genes(COL-1 and COL-3)in human dermal fibroblasts.Thereafter,biocompatibility assessment in vitro showed that the CTS/PLGA fibers per se had poorer cell adhesion and proliferation capacity than the PLGA fibers,but were cytocompatible and promoted cell migration and secretion of collagen.Moreover,after subcutaneous embedding for 2 and 4weeks in vivo,H&E staining and CD68 immumohistochemical staining results revealed that the CTS/PLGA fibers significantly reduced the recruitment of inflammatory cells,the formation of foreign body giant cells(FBGCs)and vessel structures,but promoted the penetration of fibroblasts and ECM secretion.(2)Based on the pH-neutral capability of chitosan and its advantage of chemostructural similarity to the glycosaminoglycans(GAGs)of cartilage ECM,the electrospun PLGA fibers were processed to short fibers and then incorporated into a citric acid-modified chitosan(CC)hydrogel for mechanical reinforcement and structural biomimicking,and meanwhile cartilage-decellularized matrix(CDM)was introduced for biochemical signaling to promote the chondroinduction activity.3D porous scaffolds with compositional,structural and biochemical biomimicking characteristics were fabricated through a cryogelation and lyophilization combined process,from which the chondroinduction activity and cartilage regeneration effect of the engineered highly biomimetic scaffolds were thoroughly evaluated.It was found that the incorporation of PLGA short fibers and CDM remarkably strengthened the mechanical properites of the CC hydrogel(+349% in compressive strength and +153% in Young’s modulus),which also exhibited large pore size,appropriate porosity,and fast water absorption ability.Meanwhile,the PLGA short fibers-reinforced CC scaffold(i.e.,the Fib/CC)also possessed pH-neutral capacity,which effectively alleviated the recruitment of inflammatory cells and the formation of FBGCs after subcutaneous embedding.In vitro,the engineered highly biomimetic CDM-Fib/CC scaffold significantly promoted the adhesion and proliferation of chondrocytes,and supported the formation of matured cartilage tissue with cartilage-like structure and deposition of abundant cartilage ECM-specific GAGs and type II collagen.The enhanced mechanical competency and chondroinduction capacity with the engineered CDM-Fib/CC scaffold eventually fulfilled successful in situ osteochondral regeneration in a rabbit model.(3)From the perspective of introducing anti-inflammatory biochemical signaling for scaffold functionalization,the small molecule hesperetin(Hes)with an inflammation-ameliorating function,was loaded on polydopamine(PDA)-modified electrospun aligned PLLA fibers by the adsorption effect of non-covalent(e.g.,π-π superposition and hydrogen bond)interaction.The efficacy of Hes-mediated inflammation alleviation and the influence of Hes-loading on chondrocyte function with the enginerred fibrous scaffolds were subsequently evaluated.Our results showed that Hes was successfully loaded onto the PDA-coated PLLA fibers and could also be released slowly in vitro.Moreover,the engineered Hes@PDA/PLLA scaffolds demonstrated to effectively down-regulate the expression of pro-inflammatory genes(e.g.,TNF-α,IL-1β and INOS)and up-regulate the expression of anti-inflammatory genes(e.g.,CD206 and IL-4),thus modulating the activated RAW264.7 macrophages to transit from M1 phenotype to M2 phenotype.When having the chondrocytes cultured with the Hes@PDA/PLLA scaffolds,it was found that the Hes-laden scaffolds supported the inflammation-activated chondrocytes to down-regulate the expression of pro-inflammatory genes(e.g.,TNF-α,IL-1β and INOS)and matrix metalloproteinase 3(MMP3),and promoted the cells to express cartilage ECM-specific genes(e.g.,Aggrecan,Collagen Ⅱ and Sox9)and secret the matrix components(e.g.,GAGs and collagen),beneficial for cartilage formation.To summarize,this study developed a kind of “pH-neutral” functionality fibers of CTS/PLGA with shell-core structure by coaxial electrospinning,in which the CTS-shell was employed to play the role of in situ neutralizing the acidic degradation products of PLGA-core,thus enabling to alleviate the inflammatory response for improving the long-term biocompatibility of the fibrous scaffolds.Based on the pH-neutral property of CTS and its chemostructural similarity to the GAGs,PLGA short fibers and CDM were incorporated into CTS-based hydrogel to construct highly biomimetic cartilage scaffold with enhanced mechanical properties and chondroinduction capability,thus leading to successful repair and regeneration of AC defects in vivo.Finally,by having the Hes loaded on the PDA-modified electrospun PLLA fibers,a new functional fibrous scaffold for the early inflammation-ameliorating purpose upon implantation was developed.These exploring investigations will not only enrich and deepen our understanding on the efficacy and mechanism of the engineered biomimetic scaffolds with inflammation-ameliorating function for tissue regeneration(such as cartilage tissue),but also provide new ideas for the design and construction of biomimetic scaffolds with improved biocompatibility.