Tissue engineering is rapidly progressing toward clinical application.In the musculoskeletal field,there has been an increasing necessity for bone and cartilage replacement.Despite the promising translational potentia...Tissue engineering is rapidly progressing toward clinical application.In the musculoskeletal field,there has been an increasing necessity for bone and cartilage replacement.Despite the promising translational potential of tissue engineering approaches,careful attention should be given to the quality of developed constructs to increase the real applicability to patients.After a general introduction to musculoskeletal tissue engineering,this narrative review aims to offer an overview of methods,starting from classical techniques,such as gene expression analysis and histology,to less common methods,such as Raman spectroscopy,microcomputed tomography,and biosensors,that can be employed to assess the quality of constructs in terms of viability,morphology,or matrix deposition.A particular emphasis is given to standards and good practices(GXP),which can be applicable in different sectors.Moreover,a classification of the methods into destructive,noninvasive,or conservative based on the possible further development of a preimplant quality monitoring system is proposed.Biosensors in musculoskeletal tissue engineering have not yet been used but have been proposed as a novel technology that can be exploited with numerous advantages,including minimal invasiveness,making them suitable for the development of preimplant quality control systems.展开更多
Osteochondral defects (OCD) are common but difficult to heal due to the low intrinsic repair capacity of cartilage and its complex hierarchical structure. In osteoarthritis (OA), OCD become more challenging to repair ...Osteochondral defects (OCD) are common but difficult to heal due to the low intrinsic repair capacity of cartilage and its complex hierarchical structure. In osteoarthritis (OA), OCD become more challenging to repair as both cartilage and subchondral bone regeneration are further impaired due to the arthritic environment. Numerous biomaterials have been developed and tested in osteochondral defects while ignoring the inflammatory environment. To target this challenging underlying pathophysiology, we designed and fabricated a biphasic porous and degradable scaffold incorporating anti-inflammatory and anabolic molecules by low-temperature rapid prototyping technology, and its effects on promoting osteochondral regeneration were evaluated using our well-established OA-OCD rabbit model. The biphasic porous scaffolds consisted of poly lactic-co-glycolic acid (PLGA) with kartogenin (KGN) for cartilage repair and PLGA and β-calcium phosphate (PLGA/β-TCP) with cinnamaldehyde (CIN) for subchondral bone repair. KGN is a molecule for promoting chondrogenesis and CIN is a phytomolecule for enhancing osteogenesis and alleviating inflammation. The biphasic scaffolds PLGA/KGN-PLGA/β-TCP/CIN (PK/PTC) with bio-mimic structure provided stable mechanical properties and exhibited excellent biocompatibility to support cell adhesion, proliferation, migration, and distribution. Furthermore, KGN and CIN within biphasic scaffolds could be released in a controlled and sustained mode, and the biphasic scaffold degraded slowly in vitro . Evaluating the repair of 16-weeks post-implantation into critically sized OA-OCD rabbit models revealed that the biphasic scaffold could promote subchondral bone and cartilage regeneration, as well as reverse subchondral osteosclerosis caused by inflammation in vivo . These findings support the utilization of the PK/PTC scaffold for osteochondral regeneration and provide a promising potential strategy for clinical application for the treatment of patients with OA-OCD.展开更多
Cartilage Decellularized ExtraCellular Matrix(dECM)materials have shown promising cartilage regenera-tion capacity due to their chondrogenic bioactivity.However,the limited retention of ECM components and the reduced ...Cartilage Decellularized ExtraCellular Matrix(dECM)materials have shown promising cartilage regenera-tion capacity due to their chondrogenic bioactivity.However,the limited retention of ECM components and the reduced integrity of functional ECM molecules during traditional decellularization processes im-pair the biomimicry of these materials.The current study aims to fabricate biomimetic materials con-taining decellularized cartilage particles that have an intact molecular structure and native composition as biomaterial inks and hydrogels for cartilage repair.For this,we established a novel two-fraction de-cellularization strategy for the preparation of reconstituted dECM(rdECM)particles by mixing the two-fraction components,as well as a one-fraction decellularization strategy for the preparation of biomimetic dECM(bdECM)particles.Hyaluronic acid-tyramine(THA)hydrogels containing rdECM or bdECM particles were produced and characterized via rheological test,swelling and stability evaluation,and compression test.The results showed that our novel decellularization strategies preserved intact proteoglycans and collagen at a higher retention rate with adequate DNA removal compared to traditional methods of de-cellularization.The addition of rdECM or bdECM particles significantly increased the shear moduli of the THA bioinks while preserving their shear-thinning properties.bdECM particle-embedded THA hydrogels also achieved long-term stability with a swelling ratio of 70%and high retention of glycosaminoglycans and collagen after long-term incubation,while rdECM particle-embedded THA hydrogels showed unsat-isfactory stability as self-standing biomaterials.Compared to pure THA hydrogels,the addition of bdECM particles significantly enhanced the compression moduli.In summary,our decellularization methods are successful in the retention of functional and intact cartilage components with high yield.Both rdECM and bdECM particles can be supplemented in THA bioinks for biomimetic cartilage 3D printing.Hydro-gels with cartilage bdECM particles possess the functional structure and the natural composition of car-tilage ECM,long-term stability,and enhanced mechanical properties,and are promising biomaterials for cartilage repair.展开更多
The osteogenic microenvironment of bone-repairing materials plays a key role in accelerating bone regeneration but remains incompletely defined,which significantly limits the application of such bioactive materials.He...The osteogenic microenvironment of bone-repairing materials plays a key role in accelerating bone regeneration but remains incompletely defined,which significantly limits the application of such bioactive materials.Here,the transcriptional landscapes of different osteogenic microenvironments,including three-dimensional(3D)hydroxyapatite(HA)scaffolds and osteogenic medium(OM),for mesenchymal stromal cells(MSCs)in vitro were mapped at single-cell resolution.Our findings suggested that an osteogenic process reminiscent of endochondral ossification occurred in HA scaffolds through sequential activation of osteogenic-related signaling pathways,along with inflammation and angiogenesis,but inhibition of adipogenesis and fibrosis.Moreover,we revealed the mechanism during OM-mediated osteogenesis involves the ZBTB16 and WNT signaling pathways.Heterogeneity of MSCs was also demonstrated.In vitro ossification of LRRC75A+MSCs was shown to have better utilization of WNT-related ossification process,and PCDH10+MSCs with superiority in hydroxyapatite-related osteogenic process.These findings provided further understanding of the cellular activity modulated by OM conditions and HA scaffolds,providing new insights for the improvement of osteogenic biomaterials.This atlas provides a blueprint for research on MSC heterogeneity and the osteogenic microenvironment of HA scaffolds and a database reference for the application of bioactive materials for bone regeneration.展开更多
It has been proven that the mechanical microenvironment can impact the differentiation of mesenchymal stem cells(MSCs).However,the effect of mechanical stimuli in biofabricating hydroxyapatite scaffolds on the inflamm...It has been proven that the mechanical microenvironment can impact the differentiation of mesenchymal stem cells(MSCs).However,the effect of mechanical stimuli in biofabricating hydroxyapatite scaffolds on the inflammatory response of MSCs remains unclear.This study aimed to investigate the effect of mechanical loading on the inflammatory response of MSCs seeded on scaffolds.Cyclic mechanical loading was applied to biofabricate the cell-scaffold composite for 15 min/day over 7,14,or 21 days.At the predetermined time points,culture supernatant was collected for inflammatory mediator detection,and gene expression was analyzed by qRT-PCR.The results showed that the expression of inflammatory mediators(IL1B and IL8)was downregulated(p<0.05)and the expression of ALP(p<0.01)and COL1A1(p<0.05)was upregulated under mechanical loading.The cell-scaffold composites biofabricated with or without mechanical loading were freeze-dried to prepare extracellular matrix-based scaffolds(ECM-based scaffolds).Murine macrophages were seeded on the ECM-based scaffolds to evaluate their polarization.The ECM-based scaffolds that were biofabricated with mechanical loading before freeze-drying enhanced the expression of M2 polarization-related biomarkers(Arginase 1 and Mrc1,p<0.05)of macrophages in vitro and increased bone volume/total volume ratio in vivo.Overall,these findings demonstrated that mechanical loading could dually modulate the inflammatory responses and osteogenic differentiation of MSCs.Besides,the ECM-based scaffolds that were biofabricated with mechanical loading before freeze-drying facilitated the M2 polarization of macrophages in vitro and bone regeneration in vivo.Mechanical loading may be a promising biofabrication strategy for bone biomaterials.展开更多
The depletion of chondroitin sulfates(CSs)within the intervertebral disc(IVD)during degenerative disc disease(DDD)results in a decrease in tissue hydration,a loss of fluid movement,cell apoptosis,a loss of nerve growt...The depletion of chondroitin sulfates(CSs)within the intervertebral disc(IVD)during degenerative disc disease(DDD)results in a decrease in tissue hydration,a loss of fluid movement,cell apoptosis,a loss of nerve growth inhibition and ultimately,the loss of disc function.To date,little is known with regards to the structure and content of chondroitin sulfates(CSs)during IVD ageing.The behavior of glycosaminoglycans(GAGs),specifically CSs,as well as xylosyltransferase I(XT-I)and glucuronyltransferase I(GT-I),two key enzymes involved in CS synthesis as a primer of glycosaminoglycan(GAG)chain elongation and GAG synthesis in the nucleus pulposus(NP),respectively,were evaluated in a bovine ageing IVD model.Here,we showed significant changes in the composition of GAGs during the disc ageing process(6-month-old,2-year-old and 8-year-old IVDs representing the immature to mature skeleton).The CS quantity and composition of annulus fibrosus(AF)and NP were determined.The expression of both XT-I and GT-I was detected using immunohistochemistry.A significant decrease in GAGs was observed during the ageing process.CSs are affected at both the structural and quantitative levels with important changes in sulfation observed upon maturity,which correlated with a decrease in the expression of both XT-I and GT-I.A progressive switch of the sulfation profile was noted in both NP and AF tissues from 6 months to 8 years.These changes give an appreciation of the potential impact of CSs on the disc biology and the development of therapeutic approaches for disc regeneration and repair.展开更多
文摘Tissue engineering is rapidly progressing toward clinical application.In the musculoskeletal field,there has been an increasing necessity for bone and cartilage replacement.Despite the promising translational potential of tissue engineering approaches,careful attention should be given to the quality of developed constructs to increase the real applicability to patients.After a general introduction to musculoskeletal tissue engineering,this narrative review aims to offer an overview of methods,starting from classical techniques,such as gene expression analysis and histology,to less common methods,such as Raman spectroscopy,microcomputed tomography,and biosensors,that can be employed to assess the quality of constructs in terms of viability,morphology,or matrix deposition.A particular emphasis is given to standards and good practices(GXP),which can be applicable in different sectors.Moreover,a classification of the methods into destructive,noninvasive,or conservative based on the possible further development of a preimplant quality monitoring system is proposed.Biosensors in musculoskeletal tissue engineering have not yet been used but have been proposed as a novel technology that can be exploited with numerous advantages,including minimal invasiveness,making them suitable for the development of preimplant quality control systems.
基金supported by the collaborative project from the National Key R&D Program of China and Innovation and Tech-nology Fund Mainland-Hong Kong Joint Funding Scheme(Nos.2021YFE0202300 and MHP/011/20)the Sino-Swiss collaborative project from the Ministry of Science and Technology and the Swiss National Science Foundation under the SSSTC program(Grant Nos.2015DFG32200 and 156362)+2 种基金Shenzhen Collaborative Innovation Plan-International Cooperation Project(Grant No.GJHZ20190821160803823)Development and Reform Commission of Shenzhen Municipality(2019)(No.561)Shenzhen Double Chain Project for Innovation and Development Industry supported by Bureau of Industry and Information Technology of Shenzhen(No.201908141541).
文摘Osteochondral defects (OCD) are common but difficult to heal due to the low intrinsic repair capacity of cartilage and its complex hierarchical structure. In osteoarthritis (OA), OCD become more challenging to repair as both cartilage and subchondral bone regeneration are further impaired due to the arthritic environment. Numerous biomaterials have been developed and tested in osteochondral defects while ignoring the inflammatory environment. To target this challenging underlying pathophysiology, we designed and fabricated a biphasic porous and degradable scaffold incorporating anti-inflammatory and anabolic molecules by low-temperature rapid prototyping technology, and its effects on promoting osteochondral regeneration were evaluated using our well-established OA-OCD rabbit model. The biphasic porous scaffolds consisted of poly lactic-co-glycolic acid (PLGA) with kartogenin (KGN) for cartilage repair and PLGA and β-calcium phosphate (PLGA/β-TCP) with cinnamaldehyde (CIN) for subchondral bone repair. KGN is a molecule for promoting chondrogenesis and CIN is a phytomolecule for enhancing osteogenesis and alleviating inflammation. The biphasic scaffolds PLGA/KGN-PLGA/β-TCP/CIN (PK/PTC) with bio-mimic structure provided stable mechanical properties and exhibited excellent biocompatibility to support cell adhesion, proliferation, migration, and distribution. Furthermore, KGN and CIN within biphasic scaffolds could be released in a controlled and sustained mode, and the biphasic scaffold degraded slowly in vitro . Evaluating the repair of 16-weeks post-implantation into critically sized OA-OCD rabbit models revealed that the biphasic scaffold could promote subchondral bone and cartilage regeneration, as well as reverse subchondral osteosclerosis caused by inflammation in vivo . These findings support the utilization of the PK/PTC scaffold for osteochondral regeneration and provide a promising potential strategy for clinical application for the treatment of patients with OA-OCD.
基金AO Foundation and AOSpine Inter-national.Peng Guo and Nan Jiang were funded by Sino Swiss Sci-ence and Technology Cooperation Program(Nos.EG-CN_01-032019 and EG-CN_04-042018)China Scholarship Council.MD and GM gratefully acknowledge funding from the Swiss National Sci-ence Foundation(SNSF,No.310030E_189310).
文摘Cartilage Decellularized ExtraCellular Matrix(dECM)materials have shown promising cartilage regenera-tion capacity due to their chondrogenic bioactivity.However,the limited retention of ECM components and the reduced integrity of functional ECM molecules during traditional decellularization processes im-pair the biomimicry of these materials.The current study aims to fabricate biomimetic materials con-taining decellularized cartilage particles that have an intact molecular structure and native composition as biomaterial inks and hydrogels for cartilage repair.For this,we established a novel two-fraction de-cellularization strategy for the preparation of reconstituted dECM(rdECM)particles by mixing the two-fraction components,as well as a one-fraction decellularization strategy for the preparation of biomimetic dECM(bdECM)particles.Hyaluronic acid-tyramine(THA)hydrogels containing rdECM or bdECM particles were produced and characterized via rheological test,swelling and stability evaluation,and compression test.The results showed that our novel decellularization strategies preserved intact proteoglycans and collagen at a higher retention rate with adequate DNA removal compared to traditional methods of de-cellularization.The addition of rdECM or bdECM particles significantly increased the shear moduli of the THA bioinks while preserving their shear-thinning properties.bdECM particle-embedded THA hydrogels also achieved long-term stability with a swelling ratio of 70%and high retention of glycosaminoglycans and collagen after long-term incubation,while rdECM particle-embedded THA hydrogels showed unsat-isfactory stability as self-standing biomaterials.Compared to pure THA hydrogels,the addition of bdECM particles significantly enhanced the compression moduli.In summary,our decellularization methods are successful in the retention of functional and intact cartilage components with high yield.Both rdECM and bdECM particles can be supplemented in THA bioinks for biomimetic cartilage 3D printing.Hydro-gels with cartilage bdECM particles possess the functional structure and the natural composition of car-tilage ECM,long-term stability,and enhanced mechanical properties,and are promising biomaterials for cartilage repair.
基金This study was supported by the National Key R&D Program of China(Grant no.2017YFC1105000)the National Natural Science Foundation of China(Grant no.81772400,31900583,31430030)+4 种基金the Fundamental Research Funds for the Central Universities(Grant no.19ykzd05)the Natural Science Foundation of Guangzhou City(Grant no.201704030082,201807010031)the Foundation of Shenzhen Committee for Science and Technology Innovation(Grant no.JCYJ20190809142211354,GJHZ20180929160004704)the Sanming Project of Medicine in Shenzhen(Grant no.SZSM201911002)and the Beijing Municipal Health Commission(Grant no.BMHC-2021-X,BMHC-2019-9,BMHC-2018-4,PXM2020_026275_000002).Special thanks are extended to Dr.Cheng Ruijuan for technical support.
文摘The osteogenic microenvironment of bone-repairing materials plays a key role in accelerating bone regeneration but remains incompletely defined,which significantly limits the application of such bioactive materials.Here,the transcriptional landscapes of different osteogenic microenvironments,including three-dimensional(3D)hydroxyapatite(HA)scaffolds and osteogenic medium(OM),for mesenchymal stromal cells(MSCs)in vitro were mapped at single-cell resolution.Our findings suggested that an osteogenic process reminiscent of endochondral ossification occurred in HA scaffolds through sequential activation of osteogenic-related signaling pathways,along with inflammation and angiogenesis,but inhibition of adipogenesis and fibrosis.Moreover,we revealed the mechanism during OM-mediated osteogenesis involves the ZBTB16 and WNT signaling pathways.Heterogeneity of MSCs was also demonstrated.In vitro ossification of LRRC75A+MSCs was shown to have better utilization of WNT-related ossification process,and PCDH10+MSCs with superiority in hydroxyapatite-related osteogenic process.These findings provided further understanding of the cellular activity modulated by OM conditions and HA scaffolds,providing new insights for the improvement of osteogenic biomaterials.This atlas provides a blueprint for research on MSC heterogeneity and the osteogenic microenvironment of HA scaffolds and a database reference for the application of bioactive materials for bone regeneration.
基金This research was supported by the National Natural Science Foundation of China(Grant no.32071351,81772400 and 31900583,32071341)the Fundamental Research Funds for the Central Universities(Grant no.19ykzd05)+3 种基金the Committee for Science and Technology Innovation of Shenzhen(Grant no.JCYJ20190809142211354 and GJHZ20180929160004704)the Sanming Project of Medicine in Shenzhen(Grant no.SZSM201911002)the Natural Science Foundation of Guangzhou City(Grant no.201807010031,201704030082)the Beijing Municipal Health Commission(Grant no.BMHC-2019-9,BMHC-2018-4,PXM2020_026275_000002).
文摘It has been proven that the mechanical microenvironment can impact the differentiation of mesenchymal stem cells(MSCs).However,the effect of mechanical stimuli in biofabricating hydroxyapatite scaffolds on the inflammatory response of MSCs remains unclear.This study aimed to investigate the effect of mechanical loading on the inflammatory response of MSCs seeded on scaffolds.Cyclic mechanical loading was applied to biofabricate the cell-scaffold composite for 15 min/day over 7,14,or 21 days.At the predetermined time points,culture supernatant was collected for inflammatory mediator detection,and gene expression was analyzed by qRT-PCR.The results showed that the expression of inflammatory mediators(IL1B and IL8)was downregulated(p<0.05)and the expression of ALP(p<0.01)and COL1A1(p<0.05)was upregulated under mechanical loading.The cell-scaffold composites biofabricated with or without mechanical loading were freeze-dried to prepare extracellular matrix-based scaffolds(ECM-based scaffolds).Murine macrophages were seeded on the ECM-based scaffolds to evaluate their polarization.The ECM-based scaffolds that were biofabricated with mechanical loading before freeze-drying enhanced the expression of M2 polarization-related biomarkers(Arginase 1 and Mrc1,p<0.05)of macrophages in vitro and increased bone volume/total volume ratio in vivo.Overall,these findings demonstrated that mechanical loading could dually modulate the inflammatory responses and osteogenic differentiation of MSCs.Besides,the ECM-based scaffolds that were biofabricated with mechanical loading before freeze-drying facilitated the M2 polarization of macrophages in vitro and bone regeneration in vivo.Mechanical loading may be a promising biofabrication strategy for bone biomaterials.
基金This publication has emanated from research conducted with the financial support of Science Foundation Ireland(SFI)cofunded under the European Regional Development Fund under Grant Number 13/RC/2073.
文摘The depletion of chondroitin sulfates(CSs)within the intervertebral disc(IVD)during degenerative disc disease(DDD)results in a decrease in tissue hydration,a loss of fluid movement,cell apoptosis,a loss of nerve growth inhibition and ultimately,the loss of disc function.To date,little is known with regards to the structure and content of chondroitin sulfates(CSs)during IVD ageing.The behavior of glycosaminoglycans(GAGs),specifically CSs,as well as xylosyltransferase I(XT-I)and glucuronyltransferase I(GT-I),two key enzymes involved in CS synthesis as a primer of glycosaminoglycan(GAG)chain elongation and GAG synthesis in the nucleus pulposus(NP),respectively,were evaluated in a bovine ageing IVD model.Here,we showed significant changes in the composition of GAGs during the disc ageing process(6-month-old,2-year-old and 8-year-old IVDs representing the immature to mature skeleton).The CS quantity and composition of annulus fibrosus(AF)and NP were determined.The expression of both XT-I and GT-I was detected using immunohistochemistry.A significant decrease in GAGs was observed during the ageing process.CSs are affected at both the structural and quantitative levels with important changes in sulfation observed upon maturity,which correlated with a decrease in the expression of both XT-I and GT-I.A progressive switch of the sulfation profile was noted in both NP and AF tissues from 6 months to 8 years.These changes give an appreciation of the potential impact of CSs on the disc biology and the development of therapeutic approaches for disc regeneration and repair.