Regulation Of Cartilage And Osteochondral Regeneration Based On The Microenvironment Of The Articular Cavity

Cartilage is a tissue that lacks blood vessels,neurons,and the lymphatic circulatory system.So,its regenerative capacity is restricted.The difficulty of repairing cartilage injury,particularly for articular cartilage,frequently leads to severe arthritic disease,which exacerbates the defect and resulting in a complete osteochondral defect.Clinically,palliative therapy methods,microfracture surgery,and autologous cartilage transplantation are most commonly used.However,the majority of them generate fibrocartilage,and the long-term repair impact is not optimal.As a result,tissue engineering combining biodegradable scaffolds,seed cells,and bioactive factors have steadily evolved.Regenerative cartilage is a complex and tough self-healing process.In the damaged cartilage,the hypoxic microenvironment will be disrupted,causing an inflammatory response and subchondral bone collapse.The structure and functionality of the implanted scaffolds have an impact on articular cartilage microenvironment remodeling and tissue regeneration.Targeted design of tissue engineering materials for different types of cartilage injury,as well as loading of more stable bioactive ions,using factors such as hypoxic conditions/mechanical stimulation,will aid in cartilage and osteochondral defects regeneration and osteoarthritis（OA）prevention.Mg²⁺,a bioactive ion found naturally in the body,has been recognized as an important role in osteochondral bone rebuilding.In addition to promoting cell migration,angiogenesis,and supporting the regeneration of vascularized bone tissue.Injecting Mg Cl₂ into the joint cavity can help to reduce inflammation and preserve damaged cartilage.However,the differences and mechanisms of Mg²⁺in promoting chondrogenesis and osteogenesis have not been well studied.In this work,Mg²⁺was employed as a research object,and its ability to regulate the activity of stem cells,chondrocytes,macrophages,and osteoclasts was thoroughly investigated.In addition to its potential to stimulate chondrogenic/osteogenic differentiation of stem cells at optimum concentrations.It can also work in tandem with other factors（hypoxia mimics）to improve cartilage regeneration.Furthermore,a series of structurally and functionally bionic cartilage tissue engineering scaffolds were designed and prepared in accordance with various cartilage injury models.As well as the articular cavity microenvironment,it was used to assess the scaffolds’effect on regulating bone marrow mesenchymal stem cells（BMSCs）differentiation.The proposed problems and research include:（1）A hydrogel-cryogel gradient porosity scaffold with osteochondral structural mimicry was developed.The gradient release of Mg²⁺was achieved by adding inorganic components（Magnesium basic carbonate,MCH）into the bionic cartilage layer and subchondral bone layer.At the same time,a hypoxia mimetic（Desferrioxamine,DFO）was introduced into the bionic cartilage layer to mimic the chondrocytes’hypoxic microenvironment.The effect of scaffolds in the regeneration of osteochondral defects was investigated.（2）To treat osteoarthritis-related cartilage degradation,dual-network hydrogels with covalent and dynamic bonding suited for cell proliferation were designed and manufactured.The targeted functionalized BMSCs were loaded,and the effect of Mg²⁺when combined with regularly used hypoxia mimics on reducing joint inflammation and encouraging regeneration of damaged cartilage was also studied.（3）For the cartilage layer defects,freeze-thaw cycle preparation was used to prepare ion-conducting hydrogels based on polyvinyl alcohol（PVA）and relying on strong hydrogen bonding mixed with alginate（SA）and acyl-modified gelatin with multiple carboxyl groups（Acr-Gel）.Ionic cross-linking connections were created using functional Mg²⁺.The femoral condylar cartilage defect model and the trochlea cartilage defect model were also used,with varying mechanical force stimulation.The regeneration effect of composite ion-conducting hydrogels with electrical signal stimulation on pro-chondral defects was investigated.The study’s results are briefly stated as follows:1.In vitro cellular investigations demonstrated that Mg²⁺promotes chondrogenic differentiation of BMSCs at higher concentrations（≈200 ppm）.At lower concentrations（≈100 ppm）,Mg²⁺promotes osteogenic differentiation more efficiently.Based on these findings,the doping ratio of MCH in the cartilage-mimicking hydrogel and bone-mimicking cryogel was investigated.Bilayer scaffolds with Mg²⁺gradients were created.To imitate the hypoxic environment of cartilage,a suitable amount of DFO was delivered into the cartilage-mimicking hydrogel.The scaffolds were made from methacryloyl-modified gelatin（Gel MA）.First,the conditions for the development of cryogel under various freezing temperatures were investigated（-20°C,-80°C,and-196°C）.Finally,pre-cooling at 4℃and freezing at-20℃were used to create cryogel with an average pore size of around 140μm.and a linked pore structure.Moreover,the designed cryogel has a connected macroporous structure,which has been shown to be beneficial to the subchondral bone region.The frozen cryogel was then coated with Gel MA hydrogel to form the bilayer scaffold.The longitudinally oriented pore structure generated at the interface of the cryogel and the hydrogel resembled the cartilage interlayer structure.This Mg²⁺gradient scaffold was studied by implanting it into osteochondral defects of rabbit knee joints over 12 weeks.The scaffold group with Mg²⁺gradient release and DFO for hypoxia mimicry in the cartilage-mimicking layer had the best potential to promote osteochondral regeneration.The neonatal tissue showed good mechanical stability and the highest COL-II expression,and its collagen orientation structure was the most similar to that of natural cartilage tissue.2.According to the findings of the preceding investigation,Mg²⁺and DFO can synergistically accelerate chondrogenic differentiation of BMSCs.As a result,a two-component conditioned medium containing Mg²⁺and other commonly used hypoxia mimics(Co²⁺,DMOG,and DFO)was further studied.The conditioned media containing only Mg²⁺was utilized as a control.A systematic investigation was done to investigate their synergistic effects on proliferation and chondrogenic differentiation of BMSCs.The Mg²⁺+DMOG group outperformed the other two systems in terms of cell proliferation,up-regulation of SOX9 and HIF-1α.It also promotes the production of macrophage M2 anti-inflammatory phenotype.And it also has an activation allowed for crosstalk behavior between chondrocytes and osteoclasts.It suppressed the production of osteoclasts,preventing the subchondral bone from being degraded in the OA model.Finally,BMSCs were functionalized using Mg²⁺+DMOG.And then they were loaded with phenylboronic acid-modified double-bonded hyaluronic acid（HAMA-PBA）dual-network hydrogels that exhibited good adherence and injectability.By in vitro cellular experiments,and in vivo subcutaneous ectopic chondrogenesis investigations in rats,the dual network hydrogel was found to be more effective at maintaining the cellular cartilage phenotype and cell proliferation behavior than the HAMA hydrogel.Finally,OA animal model showed that injecting Mg²⁺+DMOG aided in the early suppression of the inflammatory phase of OA and significantly reduced cartilage surface degradation.Following in vitro treatment with Mg²⁺+DMOG,BMSCs were injected into the joint cavity of the OA model via dual-network hydrogel loading.It was able to improve chondrocyte behavior through stem cell-associated paracrine function,efficiently reduced osteophyte by activating the hypoxia signaling pathway,and maintain normal joint space width.In comparison to the BMSCs group that did not receive Mg²⁺+DMOG conditioning or pretreatment,the experimental group not only demonstrated superior anti-inflammatory characteristics in vivo,it also improved the regeneration of injured cartilage.3.Taking into account the unique mechanical loading function of articular cartilage,PVA was employed as the foundation,with SA components inserted via strong hydrogen bonding.Acr-Gel was added to the scaffolds to increase their mechanical characteristics and ionic cross-linking.Simple freeze-thaw cycles produced a composite hydrogel（PSG）with good mechanical characteristics.And chelated with multifunctional divalent cations(Ca²⁺and Mg²⁺),respectively.PSG ion-conducting hydrogel with a biomimetic electrophysiological microenvironment was created through ionic bonding.PG hydrogel made of PVA and Acr-Gel was more ductile but mechanically weaker than PS hydrogels made of PVA and SA.Ion adsorption tests revealed that the PG group may bind more strongly to Mg²⁺,which has a smaller ionic radius.In addition,the PS group binds more strongly to Ca²⁺.The output electrical signal investigation demonstrated that PG-Mg had the highest output current value.This is owing to the weak ionic connection established by PG and Mg²⁺.It contains more freely movable positive and negative charges.As a result,PG-Mg generates higher-quality electrical signals.In contrast,PSG group both has the significant mechanical strength and toughness.It also had a strong current output when mechanically stimulated.To improve scaffold degradation properties while also increasing PSG mechanical properties,the gelatin components of the PSG were chemically crosslinked.PSG_c scaffold with improved mechanical characteristics was developed.Furthermore,it has good sensing properties and has no effect on its electrical signal output.A cellular experiment imitating mechanical stimulation in vitro were carried out.PSG-Mg_cwas shown to improve the expression of cartilage-associated phenotypes and hypoxia signaling pathways by increasing intracellular Mg²⁺endocytosis in response to mechanical force stimulation,in addition to releasing the functional Mg²⁺.A femoral condylar cartilage defect model was used,with higher mechanical force stimulation.In vivo studies show the ability to stimulate the electrophysiological microenvironment by stimulating the electrochemical behavior of the PSG-Mg_c ion-conducting hydrogel.This led to improved cartilage healing.It shows superior cartilage regeneration with a smooth surface of new tissue with no gaps,and phase delay analysis revealed a collagen distribution that was more compatible with the natural tissue.In summary,the combination of cartilage properties with a hypoxic microenvironment,a distinct mechanical environment,and the multifunctional Mg²⁺can be used,depending on the therapeutic goal and application scenarios.Considering these parameters when designing cartilage tissue engineering scaffolds.Strongly encourage the effective deployment of tissue engineering strategies.Provide precise and effective regenerative treatments for a variety of cartilage injuries.The findings of this dissertation are critical not only for explaining the biological processes involved in the treatment of cartilage injuries,but also for the clinical translation.It can be served as the best candidate for the next generation of cartilage tissue engineering scaffolds to improve the quality of spontaneous cartilage repair.