Robotic in situ bioprinting for cartilage tissue engineering

Articular cartilage damage caused by trauma or degenerative pathologies such as osteoarthritis can result in significant pain,mobility issues,and disability.Current surgical treatments have a limited capacity for efficacious cartilage repair,and long-term patient outcomes are not satisfying.Three-dimensional bioprinting has been used to fabricate biochemical and biophysical environments that aim to recapitulate the native microenvironment and promote tissue regeneration.However,conventional in vitro bioprinting has limitations due to the challenges associated with the fabrication and implantation of bioprinted constructs and their integration with the native cartilage tissue.In situ bioprinting is a novel strategy to directly deliver bioinks to the desired anatomical site and has the potential to overcome major shortcomings associated with conventional bioprinting.In this review,we focus on the new frontier of robotic-assisted in situ bioprinting surgical systems for cartilage regeneration.We outline existing clinical approaches and the utilization of robotic-assisted surgical systems.Handheld and robotic-assisted in situ bioprinting techniques including minimally invasive and non-invasive approaches are defined and presented.Finally,we discuss the challenges and potential future perspectives of in situ bioprinting for cartilage applications.

Functionalized Hydrogels for Articular Cartilage Tissue Engineering

Articular cartilage(AC)is an avascular and flexible connective tissue located on the bone surface in the diarthrodial joints.AC defects are common in the knees of young and physically active individuals.Because of the lack of suitable tissue-engineered artificial matrices,current therapies for AC defects,espe-cially full-thickness AC defects and osteochondral interfaces,fail to replace or regenerate damaged carti-lage adequately.With rapid research and development advancements in AC tissue engineering(ACTE),functionalized hydrogels have emerged as promising cartilage matrix substitutes because of their favor-able biomechanical properties,water content,swelling ability,cytocompatibility,biodegradability,and lubricating behaviors.They can be rationally designed and conveniently tuned to simulate the extracel-lular matrix of cartilage.This article briefly introduces the composition,structure,and function of AC and its defects,followed by a comprehensive review of the exquisite(bio)design and(bio)fabrication of func-tionalized hydrogels for AC repair.Finally,we summarize the challenges encountered in functionalized hydrogel-based strategies for ACTE both in vivo and in vitro and the future directions for clinical translation.

Recent Progress in Cartilage Tissue Engineering--Our Experience and Future Directions

Given the limited spontaneous repair that follows cartilage injury, demand is growing for tissue engi- neering approaches for cartilage regeneration. There are two major applications for tissue-engineered cartilage. One is in orthopedic surgery, in which the engineered cartilage is usually used to repair cartilage defects or loss in an articular joint or meniscus in order to restore the joint function. The other is for head and neck reconstruction, in which the engineered cartilage is usually applied to repair cartilage defects or loss in an auricle, trachea, nose, larynx, or eyelid. The challenges faced by the engineered car- tilage for one application are quite different from those faced by the engineered cartilage for the other application. As a result, the emphases of the engineering strategies to generate cartilage are usually quite different for each application. The statuses of preclinical animal investigations and of the clinical translation of engineered cartilage are also at different levels for each application. The aim of this review is to provide an opinion piece on the challenges, current developments, and future directions for cartilage engineering for both applications.

Research and progress of cartilage tissue-engineering scaffold materials

Due to the limited self healing capacity of human cartilage,the repair of defects gives rise to a challenging clinical problem.Cartilage tissue engineering provides a new method to solve cartilage repair.However,the search for a suitable biological vector material has long been the focus of research interest in this regard.In this paper,the present situation of cartilage tissue engineering vector materials is reviewed.

Self-healing poly(acrylic acid) hydrogels fabricated by hydrogen bonding and Fe^(3+) ion cross-linking for cartilage tissue engineering

Autonomous self-healing hydrogels were achieved through a dynamic combination of hydrogen bonding and ferric ion(Fe^(3+))migration.N,N′-methylenebis(acrylamide)(MBA),a cross-linking agent,was added in this study.Poly(acrylic acid)(PAA)/Fe^(3+)and PAA–MBA/Fe^(3+)hydrogels were prepared by introducing Fe^(3+)into the PAA hydrogel network.The ionic bonds were formed between Fe^(3+)ions and carboxyl groups.The microstructure,mechanical properties,and composition of hydrogels were characterized by field emission scanning electron microscopy and Fourier transform infrared spectroscopy.The experimental results showed that PAA/Fe^(3+)and PAA–MBA/Fe^(3+)hydrogels healed themselves without external stimuli.The PAA/Fe^(3+)hydrogel exhibited good mechanical properties,i.e.,the tensile strength of 50 kPa,the breaking elongation of 750%,and the self-healing efficiency of 82%.Meanwhile,the PAA–MBA/Fe^(3+)hydrogel had a tensile strength of 120 kPa.These fabricated hydrogels are biocompatible,which may have promising applications in cartilage tissue engineering.

Mechanical environment for in vitro cartilage tissue engineering assisted by in silico models

Mechanobiological study of chondrogenic cells and multipotent stem cells for articular cartilage tissue engineering(CTE)has been widely explored.The mechanical stimulation in terms of wall shear stress,hydrostatic pressure and mechanical strain has been applied in CTE in vitro.It has been found that the mechanical stimulation at a certain range can accelerate the chondrogenesis and articular cartilage tissue regeneration.This review explicitly focuses on the study of the influence of the mechanical environment on proliferation and extracellular matrix production of chondrocytes in vitro for CTE.The multidisciplinary approaches used in previous studies and the need for in silico methods to be used in parallel with in vitro methods are also discussed.The information from this review is expected to direct facial CTE research,in which mechanobiology has not been widely explored yet.

Effect of porosities of bilayered porous scaffolds on spontaneous osteochondral repair in cartilage tissue engineering

Poly(lactide-co-glycolide)-bilayered scaffolds with the same porosity or different ones on the two layers were fabricated,and the porosity effect on in vivo repairing of the osteochondral defect was examined in a comparative way for the first time.The constructs of scaffolds and bone marrow-derived mesenchymal stem cells were implanted into pre-created osteochondral defects in the femoral condyle of New Zealand white rabbits.After 12 weeks,all experimental groups exhibited good cartilage repairing according to macroscopic appearance,cross-section view,haematoxylin and eosin staining,toluidine blue staining,immunohistochemical staining and real-time polymerase chain reaction of characteristic genes.The group of 92%porosity in the cartilage layer and 77%porosity in the bone layer resulted in the best efficacy,which was understood by more biomechanical mimicking of the natural cartilage and subchondral bone.This study illustrates unambiguously that cartilage tissue engineering allows for a wide range of scaffold porosity,yet some porosity group is optimal.It is also revealed that the biomechanical matching with the natural composite tissue should be taken into consideration in the design of practical biomaterials,which is especially important for porosities of a multi-compartment scaffold concerning connected tissues.

Microcarriers in application for cartilage tissue engineering: Recent progress and challenges

Successful regeneration of cartilage tissue at a clinical scale has been a tremendous challenge in the past decades. Microcarriers (MCs), usually used for cell and drug delivery, have been studied broadly across a wide range of medical fields, especially the cartilage tissue engineering (TE). Notably, microcarrier systems provide an attractive method for regulating cell phenotype and microtissue maturations, they also serve as powerful injectable carriers and are combined with new technologies for cartilage regeneration. In this review, we introduced the typical methods to fabricate various types of microcarriers and discussed the appropriate ma-terials for microcarriers. Furthermore, we highlighted recent progress of applications and general design prin-ciple for microcarriers. Finally, we summarized the current challenges and promising prospects of microcarrier-based systems for medical applications. Overall, this review provides comprehensive and systematic guidelines for the rational design and applications of microcarriers in cartilage TE.

Mesenchymal stem cells loaded on 3D-printed gradient poly(e-caprolactone)/methacrylated alginate composite scaffolds for cartilage tissue engineering

Cartilage has limited self-repair ability due to its avascular,alymphatic and aneural features.The combination of three-dimensional(3D)printing and tissue engineering provides an up-and-coming approach to address this issue.Here,we designed and fabricated a tri-layered(superficial layer(SL),middle layer(ML)and deep layer(DL))stratified scaffold,inspired by the architecture of collagen fibers in native cartilage tissue.The scaffold was composed of 3D printed depth-dependent gradient poly(e-caprolactone)(PCL)impregnated with methacrylated alginate(ALMA),and its morphological analysis and mechanical properties were tested.To prove the feasibility of the composite scaffolds for cartilage regeneration,the viability,proliferation,collagen deposition and chondrogenic differentiation of embedded rat bone marrow mesenchymal stem cells(BMSCs)in the scaffolds were assessed by Live/dead assay,CCK-8,DNA content,cell morphology,immunofluorescence and real-time reverse transcription polymerase chain reaction.BMSCs-loaded gradient PCL/ALMA scaffolds showed excellent cell survival,cell proliferation,cell morphology,collagen II deposition and hopeful chondrogenic differentiation compared with three individual-layer scaffolds.Hence,our study demonstrates the potential use of the gradient PCL/ALMA construct for enhanced cartilage tissue engineering.

Preparation and characterization of methacrylated gelatin/bacterial cellulose composite hydrogels for cartilage tissue engineering

Methacrylated gelatin(GelMA)/bacterial cellulose(BC)composite hydrogels have been successfully prepared by immersing BC particles in GelMA solution followed by photo-crosslinking.The morphology of GelMA/BC hydrogel was examined by scanning electron microscopy and compared with pure GelMA.The hydrogels had very well interconnected porous network structure,and the pore size decreased from 200 to 10 mm with the increase of BC content.The composite hydrogels were also characterized by swelling experiment,X-ray diffraction,thermogravimetric analysis,rheology experiment and compressive test.The composite hydrogels showed significantly improved mechanical properties compared with pure GelMA.In addition,the biocompatility of composite hydrogels were preliminarily evaluated using human articular chondrocytes.The cells encapsulated within the composite hydrogels for 7 days proliferated and maintained the chondrocytic phenotype.Thus,the GelMA/BC composite hydrogels might be useful for cartilage tissue engineering.

Chondroinductive/chondroconductive peptides and their-functionalized biomaterials for cartilage tissue engineering

The repair of articular cartilage defects is still challenging in the fields of orthopedics and maxillofacial surgery due to the avascular structure of articular cartilage and the limited regenerative capacity of mature chondrocytes.To provide viable treatment options,tremendous efforts have been made to develop various chondrogenically-functionalized biomaterials for cartilage tissue engineering.Peptides that are derived from and mimic the functions of chondroconductive cartilage extracellular matrix and chondroinductive growth factors,represent a unique group of bioactive agents for chondrogenic functionalization.Since they can be chemically synthesized,peptides bear better reproducibility,more stable efficacy,higher modifiability and yielding efficiency in comparison with naturally derived biomaterials and recombinant growth factors.In this review,we summarize the current knowledge in the designs of the chondroinductive/chondroconductive peptides,the underlying molecular mechanisms and their-functionalized biomaterials for cartilage tissue engineering.We also systematically compare their in-vitro and in-vivo efficacies in inducing chondrogenesis.Our vision is to stimulate the development of novel peptides and their-functionalized biomaterials for cartilage tissue engineering.

A HYBRID SCAFFOLD OF POLY(LACTIDE-CO-GLYCOLIDE) SPONGE FILLED WITH FIBRIN GEL FOR CARTILAGE TISSUE ENGINEERING

The poly（lactide-co-glycolide）（PLGA） sponge fabricated by a gelatin porogen leaching method was filled with fibrin gel to obtain a hybrid scaffold for chondrocytes culture in vitro.The fibrin gel evenly distributed in the hybrid scaffold with visible fibrinogen fibers after drying.In vitro culture it was found that in the hybrid scaffold the chondrocytes distributed more evenly and kept a round morphology as that in the normal cartilage.Although the chondrocytes seeded in the control PLGA sponges showed similar proliferation behavior with that in the hybrid scaffolds,they were remarkably elongated,forming a fibroblast-like morphology.Moreover,a larger amount of glycosaminoglycans was secreted in the hybrid scaffolds than that in the PLGA sponges after in vitro culture of chondrocytes for 4 weeks.The results suggest that the fibrin/PLGA hybrid scaffold may be favorably applied for cartilage tissue engineering.

Adipose tissue-derived mesenchymal stem cells and chitosan/poly(vinyl alcohol)nanofibrous scaffolds for cartilage tissue engineering

Osteoarthritis(OA)has been defined as a chronic inflammatory joint disease characterized by progressive articular cartilage degeneration.Recently growing interest in regenerative medicine,using cell therapy and tissue engineering,where cellular components in combination with engineered scaffolds and bioactive materials were used to induce functional tissue regeneration.In the present study,nanofibrous scaffold based on chitosan(CS)/poly(vinyl alcohol)(PVA)were used to develop biologically functionalized biomaterial to mimic the extracellular matrix,allowing the human adipose tissue derived mesenchymal stem cells(ADSCs)to proliferate and differentiate to chondrogenic cells.The morphology of the nanofibrous mat was examined using field emission scanning electron microscope(FE/SEM).The characteristic functional groups and the nature of the chemical bonds between atoms were evaluated using Fourier transform infrared spectroscopy(FTIR)spectrum.Characterization of the seeded cells was morphologically evaluated by scanning electron microscopy and by flow cytometry for the expression of the stem cell surface markers.The differentiation potential was verified after chondrogenic induction by analyzing the expression of chondrogenic marker genes using real-time(RT PCR).Current study suggest significant potential for the use of ADSCs with the nanofibrous scaffolds in improving the osteoarthritis pathology.

Cells from Cow Knee Joints Used to Grow New Cartilage Tissue in Laboratory

In an effort to develop a method for cartilage tissue engineering,researchers successfully used cartilage cells from cow knee joints.By creating a successful method with conditions conducive to growing healthy cartilage tissue,the findings could help lead to a new treatment cure for osteoarthritis using stem cell-based tissue engineering.Articular cartilage is tissue that is found on all the joint surfaces in the body.Since the tissue

Cartilage and facial muscle tissue engineering and regeneration： a mini review

Cartilage and facial muscle tissue provide basic yet vital functions for homeostasis throughout the body, making human survival and function highly dependent upon these somatic components. When cartilage and facial muscle tissues are harmed or completely destroyed due to disease, trauma, or any other degenerative process, homeostasis and basic body functions consequently become negatively affected. Although most cartilage and cells can regenerate themselves after any form of the aforementioned degenerative disease or trauma, the highly specific characteristics of facial muscles and the specific structures of the cells and tissues required for the proper function cannot be exactly replicated by the body itself. Thus, some form of cartilage and bone tissue engineering is necessary for proper regeneration and function. The use of progenitor cells for this purpose would be very beneficial due to their highly adaptable capabilities, as well as their ability to utilize a high diffusion rate, making them ideal for the specific nature and functions of cartilage and facial muscle tissue. Going along with this, once the progenitor cells are obtained, applying them to a scaffold within the oral cavity in the affected location allows them to adapt to the environment and create cartilage or facial muscle tissue that is specific to the form and function of the area. The principal function of the cartilage and tissue is vascularization, which requires a specific form that allows them to aid the proper flow of bodily functions related to the oral cavity such as oxygen flow and removal of waste. Facial muscle is also very thin, making its reproduction much more possible. Taking all these into consideration, this review aims to highlight and expand upon the primary benefits of the cartilage and facial muscle tissue engineering and regeneration, focusing on how these processes are performed outside of and within the body.

In vitro and In vivo Evaluation of the Developed PLGA/HAp/Zein Scaffolds for Bone-Cartilage Interface Regeneration

Objective To investigate the effect of electronspun PLGA/HAp/Zein scaffolds on the repair of cartilage defects. Methods The PLGA/HAp/Zein composite scaffolds were fabricated by electrospinning method. The physiochemical properties and biocompatibility of the scaffolds were separately characterized by scanning electron microscope （SEM）, transmission electron microscope （TEM）, and fourier transform infrared spectroscopy （FTIR）, human umbilical cord mesenchymal stem cells （hUC-MSCs） culture and animal experiments. Results The prepared PLGA/HAp/Zein scaffolds showed fibrous structure with homogenous distribution, hUC-MSCs could attach to and grow well on PLGA/HAp/Zein scaffolds, and there was no significant difference between cell proliferation on scaffolds and that without scaffolds （P〉0.05）. The PLGA/HAp/Zein scaffolds possessed excellent ability to promote in vivo cartilage formation. Moreover, there was a large amount of immature chondrocytes and matrix with cartilage lacuna on PLGA/HAp/Zein scaffolds. Conclusion The data suggest that the PLGA/HAp/Zein scaffolds possess good biocompatibility, which are anticipated to be potentially applied in cartilage tissue engineering and reconstruction.

Progress and prospect of technical and regulatory challenges on tissue-engineered cartilage as therapeutic combination product

Hyaline cartilage plays a critical role in maintaining joint function and pain.However,the lack of blood supply,nerves,and lymphatic vessels greatly limited the self-repair and regeneration of damaged cartilage,giving rise to various tricky issues in medicine.In the past 30 years,numerous treatment techniques and commercial products have been developed and practiced in the clinic for promoting defected cartilage repair and regeneration.Here,the current therapies and their relevant advantages and disadvantages will be summarized,particularly the tissue engineering strategies.Furthermore,the fabrication of tissue-engineered cartilage under research or in the clinic was discussed based on the traid of tissue engineering,that is the materials,seed cells,and bioactive factors.Finally,the commercialized cartilage repair products were listed and the regulatory issues and challenges of tissue-engineered cartilage repair products and clinical application would be reviewed.

Articular cartilage and osteochondral tissue engineering techniques:Recent advances and challenges

In spite of the considerable achievements in the field of regenerative medicine in the past several decades,osteochondral defect regeneration remains a challenging issue among diseases in the musculoskeletal system because of the spatial complexity of osteochondral units in composition,structure and functions.In order to repair the hierarchical tissue involving different layers of articular cartilage,cartilage-bone interface and subchondral bone,traditional clinical treatments including palliative and reparative methods have showed certain improvement in pain relief and defect filling.It is the development of tissue engineering that has provided more promising results in regenerating neo-tissues with comparable compositional,structural and functional characteristics to the native osteochondral tissues.Here in this review,some basic knowledge of the osteochondral units including the anatomical structure and composition,the defect classification and clinical treatments will be first introduced.Then we will highlight the recent progress in osteochondral tissue engineering from perspectives of scaffold design,cell encapsulation and signaling factor incorporation including bioreactor application.Clinical products for osteochondral defect repair will be analyzed and summarized later.Moreover,we will discuss the current obstacles and future directions to regenerate the damaged osteochondral tissues.

Enhancing cartilage repair with optimized supramolecular hydrogel-based scaffold and pulsed electromagnetic field

Functional tissue engineering strategies provide innovative approach for the repair and regeneration of damaged cartilage.Hydrogel is widely used because it could provide rapid defect filling and proper structure support,and is biocompatible for cell aggregation and matrix deposition.Efforts have been made to seek suitable scaffolds for cartilage tissue engineering.Here Alg-DA/Ac-β-CD/gelatin hydrogel was designed with the features of physical and chemical multiple crosslinking and self-healing properties.Gelation time,swelling ratio,biodegradability and biocompatibility of the hydrogels were systematically characterized,and the injectable self-healing adhesive hydrogel were demonstrated to exhibit ideal properties for cartilage repair.Furthermore,the new hydrogel design introduces a pre-gel state before photo-crosslinking,where increased viscosity and decreased fluidity allow the gel to remain in a semi-solid condition.This granted multiple administration routes to the hydrogels,which brings hydrogels the ability to adapt to complex clinical situations.Pulsed electromagnetic fields(PEMF)have been recognized as a promising solution to various health problems owing to their noninvasive properties and therapeutic potentials.PEMF treatment offers a better clinical outcome with fewer,if any,side effects,and wildly used in musculoskeletal tissue repair.Thereby we propose PEMF as an effective biophysical stimulation to be 4th key element in cartilage tissue engineering.In this study,the as-prepared Alg-DA/Ac-β-CD/gelatin hydrogels were utilized in the rat osteochondral defect model,and the potential application of PEMF in cartilage tissue engineering were investigated.PEMF treatment were proven to enhance the quality of engineered chondrogenic constructs in vitro,and facilitate chondrogenesis and cartilage repair in vivo.All of the results suggested that with the injectable self-healing adhesive hydrogel and PEMF treatment,this newly proposed tissue engineering strategy revealed superior clinical potential for cartilage defect treatment.