Optimal Construction Of Bioactive Engineered Ligament For Enhancing Graft—Bone Osseointegration

BackgroundAnterior cruciate ligament(ACL)injury is one of the most common sports injuries and surgical reconstruction is the standard of clinical treatment.Artificial ligament graft(LARS ligament made of PET)is one of the available grafts for ACL reconstruction.However,lack of osteogenic activity leads to absence of osseointegration between graft and host bone and the hyperplasia of the fibrous scar interface,consequently causing implant loosening and reconstruction failure.Therefore,it is urgent and significant to optimize ligament graft-bone integration,improve the reconstruction effect and prolong the service duration.In recent years,calcium and phosphorus materials,nanoparticles,cytokines and immunoregulatory molecules are the hot topics in the research field of bone regeneration.The modification of artificial grafts with bioactive substances to enhance the osteogenic activity of grafts is an effective method to promote graft-bone integration.In this study,bioactive engineered ligaments were constructed by optimizing the introduction of bioactive ingredients,endowing engineered ligaments with osteogenic activity,promoting new bone regeneration on the graft surface and within the graft,and resulting in graft-bone osseointegration,which aims at improving the effect of artificial ligaments reconstructing ACL.Objective1)to develop a bioactive injectable CPC/ lithium nano-hydrogel and investigate its enhancement of graft-bone integration;2)to construct artificial ligament-bone integrative grafts and verify its promoting effect on graft-bone integration;3)to design CS/BMP-2-comodified immunoregulatory engineered ligaments and study its enhancing effect on graft-bone integration;Methods1.Using synthetic nanomaterials Laponite(LP)and calcium phosphate cement(CPC)as bioactive components,LP-CPC gel and LP gel were prepared by mixing LP and CPC with gelatin solution in "one-step method".SEM,EDS and XRD were used to characterize the surface morphology and chemical composition of the gels and gelartificial ligament complex.Rabbit bone marrow mesenchymal stem cells(BMSCs)were isolated and cultured in vitro.BMSCs were co-cultured with LP/CPC gel and gelartificial ligament complex.CCK-8 test,live/dead staining and PCR test were performed to investigate the cytocompatibility and osteogenic activity of the gel materials.In vivo,artificial ligament,LP gel-artificial ligament and LP/CPC gelartificial ligament were implanted into bone tunnel in a rabbit model.After sacrificed in 1,2 and 3 months postoperatively,the samples were collected and examined using micro-CT,biomechanics test,HE and Masson staining to investigate the effect of bioactive injectable CPC/LP nano-hydrogel on graft-bone osseointegration.2.CPC was selected as the osteogenic component,and the artificial bone PDTC was prepared by polylactate carbonate(PDT)and CPC.Artificial ligament-bone integrative grafts were constructed with artificial ligament and PDTC by controllable thermocompression.SEM,EDS,XRD and biomechanics were used to characterize the materials.Rabbit BMSCs were co-cultured with PDTC and integrated grafts in vitro to detect their osteogenic activity using living/dead staining,PCR,immunofluorescence staining,ALP staining and alizarin red staining.In vivo artificial ligament grafts(ALG),artificial ligament-PDT integrative grafts(ALPG),artificial ligaments-PDTC integrative grafts(ALBG)were inplanted into bone tunnel in a rabbit model.After sacrificed at 1,3 and 6 months after surgery,the samples were examined using microCT,HE and Masson staining,VG staining,sequential fluorescent tags and biomechanical testing,to investigate the effect of integrative graft-bone integration.3.Chondroitin sulfate(CS)and BMP-2 were selected as the active components.Dopamine-modified artificial ligament was prepared as a control(p PET).The CS-PDA modified artificial ligament(C-p PET)was prepared by chemical grafting of CS.BMP-2/PDA modified artificial ligament(B/p PET)and BMP-2/CS-PDA modified artificial ligament(B/C-p PET)were prepared by physical adsorption of BMP-2.SEM,EDS,AFM and XRD were used for characterization.Four kinds of grafts were co-cultured with rat BMSCs and mouse RAW264.7 macrophages,and the immunoregulatory function and osteogenic activity of B/C-p PET were investigated by immunofluorescence staining,flow cytometry,transwell,western-blot,ALP staining and alizarin red staining.in vivo,p PET,C-p PET,B/p PET,B/C-p PET were implanted into bone tunnel in a rat model.After sacrificed at 1,2,3 months after surgery,the samples were examined using ELISA,micro-CT,HE and Masson staining,immunohistochemical and immunofluorescencal staining,sequential fluorescent tags,to validate the effect of artificial ligament-bone integration.Results1.LP gel and LP/CPC gel have good injectability and cytocompatibility,which increase the surface roughness of artificial ligament and improve its osteogenic activity by promoting the expression of osteogenic marker genes.Micro-CT results showed that bone regeneration was obviously enhanced in the LP/CPC gel-artificial ligament group.Biomechanical results show that the LAP/CPC gel significantly improved the graftbone biomechanical properties,HE staining showed LP/CPC gel significantly promoted bone regeneration around and within the grafts.Masson staining showed LAP/CPC gel can promote ligament graft-host bone integration at the graft surface,and graft-new bone integration within the grafts.2.PDTC artificial bone has good cytocompatibility and osteogenic activity,and the artificial ligament-bone integrative graft(ALBG)has the advantages of high bonding strength and flexible texture.In vitro results showed that ALBG can promote the proliferation,migration and adhesion of BMSCs,promote the expression of ALP,Osx,OCN and OPN,and increase the formation of calcium nodules.Micro-CT results showed a significant increase of new bone regeneration in the ALBG group,and biomechanical tests showed that PDTC significantly increased the biomechanical properties of integrative graft in vivo.HE staining showed a significant increase of the formation of new bone on the surface and inside the integrative graft.Masson stain demonstrated the osseointegration of of ligament graft-bone in the ALBG group.3.B/C-p PET was prepared by chemical grafting of CS and physical adsorption of BMP-2.In vitro experiments showed that C-p PET could regulate the polarization of macrophages to the M2 phenotype and promote the secretion of anti-inflammatory factors.The immunoregulatory function of CS and BMP-2 can synergistically promote osteogenic differentiation of BMSCs and up-regulate the expression of ALP,Runx2,OCN and COL I.The new bone regeneration of the B/p PET group was significantly enhanced from 1 to 2 months,while the new bone regeneration of the C-p PET group was significantly enhanced from 2 to 3 months.B/C-p PET promoted new bone formation from 1 to 3 months to obtain the best graft-bone osseous integration.Conclusions1.Bioactive injectable LP/CPC gel has high osteogenic activity,and LP/CPC nanogel significantly enhanced graft-bone osseous integration.Therefore,endowing the artificial ligament with osteogenic activity by introducing osteogenic material is a feasible way to promote the integration between the ligament graft and bone.2.The artificial ligament-bone integrative grafts complished the high-strength combination of artificial ligament and artificial bone,significantly improved the graftbone integration,and improved the biomechanical properties of ligament grafts in vivo.High-strength artificial ligament-bone graft has potential clinical value.3.B/C-p PET obtained the optimal graft-bone integration through the sequence and synergistic effect of bone regeneration induced by CS immune osteogenesis and BMP-2.Immune-regulated artificial ligaments are a new way to achieve graft-bone integration and have potential clinical value in improving the effect of ACL reconstruction.4.In this study,we firstly introduced sufficient and effective active materials to construct bioactive engineered ligament graft,and preliminarily demonstrated the promotion effect of bioactive ligament grafts on graft-bone osseointegration.Then,to solving the problem of active material shedding,the high-strength combination of the active artificial bone and the artificial ligament was fabricated to achieving the stable and sufficient introduction of active material.The artificial ligament-bone integration graft was constructed to promote the graft-bone integration.Next,immunoactive ingredients were introduced to construct immunomodulatory engineered ligaments,which inhibited the proliferation of fibrous tissue by regulating the immune microenvironment in the bone canal,gave full play to the osteogenic function of the active factors,and promoted the graft-bone integration.On the whole,this study shows that the construction of bioactive engineered ligament graft is a feasible method to solve the problem of graft-bone integration and improve the effect of ligament reconstruction.