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Dermis-isolated adult stem (DIAS) cells, abundantly available, are attractive for regenerative medicine. Strategies have been devised to isolate and to chondroinduce DIAS cells from various animals. This study aimed to characterize DIAS cells from human abdominal skin (human dermis-isolated adult stem [hDIAS] cells) and to compare and to refine various chondroinduction regimens to form functional neocartilage constructs. The stemness of hDIAS cells was verified (Phase I), three chondroinduction pretreatments were compared (Phase II), and, from these, one regimen was carried forward for refinement in Phase III for improving the mechanical properties of hDIAS cell-derived constructs. Multilineage differentiation and mesenchymal stem cell markers were observed. Among various chondroinduction pretreatments, the nodule formation pretreatment yielded constructs at least 72% larger in diameter, with higher glycosaminoglycan (GAG) content by 44%, compared with other pretreatments. Furthermore, it was found that culturing cells on nontissue culture-treated surfaces yielded constructs (1) on par with constructs derived from aggrecan-coated surfaces and (2) with superior mechanical properties than constructs derived from cells cultured on tissue culture-treated surfaces. After the nodule formation pretreatment, combined supplementation of TGF-β1, IGF-I, and fetal bovine serum significantly enhanced aggregate modulus and shear modulus by 75% and 69%, respectively, over the supplementation by TGF-β1 alone. In summary, human skin-derived DIAS cells are responsive to chondroinduction for forming neocartilage. Furthermore, the mechanical properties of the resultant human constructs can be improved by treatments shown to be efficacious in animal models. Advances made toward tissue-engineering cartilage using animal cells were shown to be applicable to hDIAS cells for cartilage repair and regeneration.

Dermis-isolated adult stem (DIAS) cells, abundantly available, are attractive for regenerative medicine. Strategies have been devised to isolate and to chondroinduce DIAS cells from various animals. This study aimed to characterize DIAS cells from human abdominal skin (human dermis-isolated adult stem [hDIAS] cells) and to compare and to refine various chondroinduction regimens to form functional neocartilage constructs. The stemness of hDIAS cells was verified (Phase I), three chondroinduction pretreatments were compared (Phase II), and, from these, one regimen was carried forward for refinement in Phase III for improving the mechanical properties of hDIAS cell-derived constructs. Multilineage differentiation and mesenchymal stem cell markers were observed. Among various chondroinduction pretreatments, the nodule formation pretreatment yielded constructs at least 72% larger in diameter, with higher glycosaminoglycan (GAG) content by 44%, compared with other pretreatments. Furthermore, it was found that culturing cells on nontissue culture-treated surfaces yielded constructs (1) on par with constructs derived from aggrecan-coated surfaces and (2) with superior mechanical properties than constructs derived from cells cultured on tissue culture-treated surfaces. After the nodule formation pretreatment, combined supplementation of TGF-β1, IGF-I, and fetal bovine serum significantly enhanced aggregate modulus and shear modulus by 75% and 69%, respectively, over the supplementation by TGF-β1 alone. In summary, human skin-derived DIAS cells are responsive to chondroinduction for forming neocartilage. Furthermore, the mechanical properties of the resultant human constructs can be improved by treatments shown to be efficacious in animal models. Advances made toward tissue-engineering cartilage using animal cells were shown to be applicable to hDIAS cells for cartilage repair and regeneration.

Tissue engineering aims to create implantable biomaterials for the repair and regeneration of damaged tissues. In vitro tissue engineering is generally based on static culture, which limits access to nutrients and lacks mechanical signaling. Using shear stress is controversial because in some cases it can lead to cell death while in others it promotes tissue regeneration. To understand how shear stress works and how it may be used to improve neotissue function, a series of studies were performed. First, a tunable device was designed to determine optimal levels of shear stress for neotissue formation. Then, computational fluid dynamics modeling showed the device applies fluid-induced shear (FIS) stress spanning three orders of magnitude on tissue-engineered cartilage (neocartilage). A beneficial window of FIS stress was subsequently identified, resulting in up to 3.6-fold improvements in mechanical properties of neocartilage in vitro. In vivo, neocartilage matured as evidenced by the doubling of collagen content toward native values. Translation of FIS stress to human derived neocartilage was then demonstrated, yielding analogous improvements in mechanical properties, such as 168% increase in tensile modulus. To gain an understanding of the beneficial roles of FIS stress, a mechanistic study was performed revealing a mechanically gated complex on the primary cilia of chondrocytes that is activated by FIS stress. This series of studies places FIS stress into the arena as a meaningful mechanical stimulation strategy for creating robust and translatable neotissues, and demonstrates the ease of incorporating FIS stress in tissue culture.

Tissue engineering aims to create implantable biomaterials for the repair and regeneration of damaged tissues. In vitro tissue engineering is generally based on static culture, which limits access to nutrients and lacks mechanical signaling. Using shear stress is controversial because in some cases it can lead to cell death while in others it promotes tissue regeneration. To understand how shear stress works and how it may be used to improve neotissue function, a series of studies were performed. First, a tunable device was designed to determine optimal levels of shear stress for neotissue formation. Then, computational fluid dynamics modeling showed the device applies fluid-induced shear (FIS) stress spanning three orders of magnitude on tissue-engineered cartilage (neocartilage). A beneficial window of FIS stress was subsequently identified, resulting in up to 3.6-fold improvements in mechanical properties of neocartilage in vitro. In vivo, neocartilage matured as evidenced by the doubling of collagen content toward native values. Translation of FIS stress to human derived neocartilage was then demonstrated, yielding analogous improvements in mechanical properties, such as 168% increase in tensile modulus. To gain an understanding of the beneficial roles of FIS stress, a mechanistic study was performed revealing a mechanically gated complex on the primary cilia of chondrocytes that is activated by FIS stress. This series of studies places FIS stress into the arena as a meaningful mechanical stimulation strategy for creating robust and translatable neotissues, and demonstrates the ease of incorporating FIS stress in tissue culture.

The zygapophysial joint, a diarthrodial joint commonly referred to as the facet joint, plays a pivotal role in back pain, a condition that has been a leading cause of global disability since 1990. Along with the intervertebral disc, the facet joint supports spinal motion and aids in spinal stability. Highly susceptible to early development of osteoarthritis, the facet is responsible for a significant amount of pain in the low-back, mid-back, and neck regions. Current noninvasive treatments cannot offer long-term pain relief, while invasive treatments can relieve pain but fail to preserve joint functionality. This review presents an overview of the facet in terms of its anatomy, functional properties, problems, and current management strategies. Furthermore, this review introduces the potential for regeneration of the facet and particular engineering strategies that could be employed as a long-term treatment.

The zygapophysial joint, a diarthrodial joint commonly referred to as the facet joint, plays a pivotal role in back pain, a condition that has been a leading cause of global disability since 1990. Along with the intervertebral disc, the facet joint supports spinal motion and aids in spinal stability. Highly susceptible to early development of osteoarthritis, the facet is responsible for a significant amount of pain in the low-back, mid-back, and neck regions. Current noninvasive treatments cannot offer long-term pain relief, while invasive treatments can relieve pain but fail to preserve joint functionality. This review presents an overview of the facet in terms of its anatomy, functional properties, problems, and current management strategies. Furthermore, this review introduces the potential for regeneration of the facet and particular engineering strategies that could be employed as a long-term treatment.

Chondral lesions frequently occur in different topographic locations of the knee. This study evaluated the functional properties among the articulating surfaces of the tibiofemoral and patellofemoral joints, and whether neo-cartilage engineered using chondrocytes from different knee locations would reflect these differences. The biomechanical properties of bovine cartilage isolated from eight locations within the tibiofemoral (medial and lateral condyle, medial and lateral tibial plateau) and patellofemoral joints (medial and lateral trochlea, medial and lateral patella) were examined. Tensile Young's moduli (tensile moduli) and aggregate moduli of the medial condyle were lower than those of the medial tibial plateau (6.11 ± 0.89 MPa vs. 7.19 ± 1.05 MPa, p = 0.04 and 354.4 ± 38.3 kPa vs. 419.4 ± 31.3 kPa, p = 0.002, respectively). Patella tensile and compressive moduli were lower than the trochlea (4.79 ± 2.01 MPa vs. 6.91 ± 2.46 MPa, p = 0.01 and 337.4 ± 37.2 kPa vs. 389.1 ± 38.3 kPa, p = 0.0005, respectively). Furthermore, chondrocytes from the above locations were used to engineer neo-cartilage, and its respective properties were evaluated. In neo-cartilage, medial condyle tensile and aggregate moduli were lower than in the medial tibial plateau (0.96 ± 0.23 MPa vs. 1.31 ± 0.31 MPa, p = 0.02, and 115.8 ± 26.0 kPa vs. 160.8 ± 18.8 kPa, p = 0.001, respectively). Compared to trochlear chondrocytes, neo-cartilage formed from patellar chondrocytes exhibited lower tensile and compressive moduli (1.16 ± 0.27 MPa vs. 0.74 ± 0.25 MPa, p

Chondral lesions frequently occur in different topographic locations of the knee. This study evaluated the functional properties among the articulating surfaces of the tibiofemoral and patellofemoral joints, and whether neo-cartilage engineered using chondrocytes from different knee locations would reflect these differences. The biomechanical properties of bovine cartilage isolated from eight locations within the tibiofemoral (medial and lateral condyle, medial and lateral tibial plateau) and patellofemoral joints (medial and lateral trochlea, medial and lateral patella) were examined. Tensile Young's moduli (tensile moduli) and aggregate moduli of the medial condyle were lower than those of the medial tibial plateau (6.11 ± 0.89 MPa vs. 7.19 ± 1.05 MPa, p = 0.04 and 354.4 ± 38.3 kPa vs. 419.4 ± 31.3 kPa, p = 0.002, respectively). Patella tensile and compressive moduli were lower than the trochlea (4.79 ± 2.01 MPa vs. 6.91 ± 2.46 MPa, p = 0.01 and 337.4 ± 37.2 kPa vs. 389.1 ± 38.3 kPa, p = 0.0005, respectively). Furthermore, chondrocytes from the above locations were used to engineer neo-cartilage, and its respective properties were evaluated. In neo-cartilage, medial condyle tensile and aggregate moduli were lower than in the medial tibial plateau (0.96 ± 0.23 MPa vs. 1.31 ± 0.31 MPa, p = 0.02, and 115.8 ± 26.0 kPa vs. 160.8 ± 18.8 kPa, p = 0.001, respectively). Compared to trochlear chondrocytes, neo-cartilage formed from patellar chondrocytes exhibited lower tensile and compressive moduli (1.16 ± 0.27 MPa vs. 0.74 ± 0.25 MPa, p

Scaffold-free systems have emerged as viable approaches for engineering load-bearing tissues. However, the tensile properties of engineered tissues have remained far below the values for native tissue. Here, by using self-assembled articular cartilage as a model to examine the effects of intermittent and continuous tension stimulation on tissue formation, we show that the application of tension alone, or in combination with matrix remodelling and synthesis agents, leads to neocartilage with tensile properties approaching those of native tissue. Implantation of tension-stimulated tissues results in neotissues that are morphologically reminiscent of native cartilage. We also show that tension stimulation can be translated to a human cell source to generate anisotropic human neocartilage with enhanced tensile properties. Tension stimulation, which results in nearly sixfold improvements in tensile properties over unstimulated controls, may allow the engineering of mechanically robust biological replacements of native tissue.

Abundance and accessibility render skin-derived stem cells an attractive cell source for tissue engineering applications. Toward assessing their utility, the variability of constructs engineered from human dermis-isolated adult stem (hDIAS) cells was examined with respect to different anatomical locations (foreskin, breast, and abdominal skin), both in vitro and in a subcutaneous, athymic mouse model. All anatomical locations yielded hDIAS cells with multi-lineage differentiation potentials, though adipogenesis was not seen for foreskin-derived hDIAS cells. Using engineered cartilage as a model, tissue engineered constructs from hDIAS cells were compared. Construct morphology differed by location. The mechanical properties of human foreskin- and abdominal skin-derived constructs were similar at implantation, remaining comparable after 4 additional weeks of culture in vivo. Breast skin-derived constructs were not mechanically testable. For all groups, no signs of abnormality were observed in the host. Addition of aggregate redifferentiation culture prior to construct formation improved chondrogenic differentiation of foreskin-derived hDIAS cells, as evident by increases in glycosaminoglycan and collagen contents. More robust Alcian blue staining and homogeneous cell populations were also observed compared to controls. Human DIAS cells elicited no adverse host responses, reacted positively to chondrogenic regimens, and possessed multi-lineage differentiation potential with the caveat that efficacy may differ by anatomical origin of the skin. Taken together, these results suggest that hDIAS cells hold promise as a potential cell source for a number of tissue engineering applications.

ObjectiveThe application of cell-based therapies in regenerative medicine is hindered by the difficulty of acquiring adequate numbers of competent cells. For the knee meniscus in particular, this may be solved by harvesting tissue from neighboring tendons and ligaments. In this study, we have investigated the potential of cells from tendon and ligament, as compared to meniscus cells, to engineer scaffold-free self-assembling fibrocartilage.MethodSelf-assembling meniscus-shaped constructs engineered from a co-culture of articular chondrocytes and either meniscus, tendon, or ligament cells were cultured for 4 weeks with TGF-β1 in serum-free media. After culture, constructs were assessed for their mechanical properties, histological staining, gross appearance, and biochemical composition including cross-link content. Correlations were performed to evaluate relationships between biochemical content and mechanical properties.ResultsIn terms of mechanical properties as well as biochemical content, constructs engineered using tenocytes and ligament fibrocytes were found to be equivalent or superior to constructs engineered using meniscus cells. Furthermore, cross-link content was found to be correlated with engineered tissue tensile properties.ConclusionTenocytes and ligament fibrocytes represent viable cell sources for engineering meniscus fibrocartilage using the self-assembling process. Due to greater cross-link content, fibrocartilage engineered with tenocytes and ligament fibrocytes may maintain greater tensile properties than fibrocartilage engineered with meniscus cells.

ObjectiveThe application of cell-based therapies in regenerative medicine is hindered by the difficulty of acquiring adequate numbers of competent cells. For the knee meniscus in particular, this may be solved by harvesting tissue from neighboring tendons and ligaments. In this study, we have investigated the potential of cells from tendon and ligament, as compared to meniscus cells, to engineer scaffold-free self-assembling fibrocartilage.MethodSelf-assembling meniscus-shaped constructs engineered from a co-culture of articular chondrocytes and either meniscus, tendon, or ligament cells were cultured for 4 weeks with TGF-β1 in serum-free media. After culture, constructs were assessed for their mechanical properties, histological staining, gross appearance, and biochemical composition including cross-link content. Correlations were performed to evaluate relationships between biochemical content and mechanical properties.ResultsIn terms of mechanical properties as well as biochemical content, constructs engineered using tenocytes and ligament fibrocytes were found to be equivalent or superior to constructs engineered using meniscus cells. Furthermore, cross-link content was found to be correlated with engineered tissue tensile properties.ConclusionTenocytes and ligament fibrocytes represent viable cell sources for engineering meniscus fibrocartilage using the self-assembling process. Due to greater cross-link content, fibrocartilage engineered with tenocytes and ligament fibrocytes may maintain greater tensile properties than fibrocartilage engineered with meniscus cells.

Stem cells are the future in tissue engineering and regeneration. In a co-culture, stem cells not only provide a target cell source with multipotent differentiation capacity, but can also act as assisting cells that promote tissue homeostasis, metabolism, growth and repair. Their incorporation into co-culture systems seems to be important in the creation of complex tissues or organs. In this review, critical aspects of stem cell use in co-culture systems are discussed. Direct and indirect co-culture methodologies used in tissue engineering are described, along with various characteristics of cellular interactions in these systems. Direct cell-cell contact, cell-extracellular matrix interaction and signalling via soluble factors are presented. The advantages of stem cell co-culture strategies and their applications in tissue engineering and regenerative medicine are portrayed through specific examples for several tissues, including orthopaedic soft tissues, bone, heart, vasculature, lung, kidney, liver and nerve. A concise review of the progress and the lessons learned are provided, with a focus on recent developments and their implications. It is hoped that knowledge developed from one tissue can be translated to other tissues. Finally, we address challenges in tissue engineering and regenerative medicine that can potentially be overcome via employing strategies for stem cell co-culture use.

Stem cells are the future in tissue engineering and regeneration. In a co-culture, stem cells not only provide a target cell source with multipotent differentiation capacity, but can also act as assisting cells that promote tissue homeostasis, metabolism, growth and repair. Their incorporation into co-culture systems seems to be important in the creation of complex tissues or organs. In this review, critical aspects of stem cell use in co-culture systems are discussed. Direct and indirect co-culture methodologies used in tissue engineering are described, along with various characteristics of cellular interactions in these systems. Direct cell-cell contact, cell-extracellular matrix interaction and signalling via soluble factors are presented. The advantages of stem cell co-culture strategies and their applications in tissue engineering and regenerative medicine are portrayed through specific examples for several tissues, including orthopaedic soft tissues, bone, heart, vasculature, lung, kidney, liver and nerve. A concise review of the progress and the lessons learned are provided, with a focus on recent developments and their implications. It is hoped that knowledge developed from one tissue can be translated to other tissues. Finally, we address challenges in tissue engineering and regenerative medicine that can potentially be overcome via employing strategies for stem cell co-culture use.

Stem cells are the future in tissue engineering and regeneration. In a co-culture, stem cells not only provide a target cell source with multipotent differentiation capacity, but can also act as assisting cells that promote tissue homeostasis, metabolism, growth and repair. Their incorporation into co-culture systems seems to be important in the creation of complex tissues or organs. In this review, critical aspects of stem cell use in co-culture systems are discussed. Direct and indirect co-culture methodologies used in tissue engineering are described, along with various characteristics of cellular interactions in these systems. Direct cell-cell contact, cell-extracellular matrix interaction and signalling via soluble factors are presented. The advantages of stem cell co-culture strategies and their applications in tissue engineering and regenerative medicine are portrayed through specific examples for several tissues, including orthopaedic soft tissues, bone, heart, vasculature, lung, kidney, liver and nerve. A concise review of the progress and the lessons learned are provided, with a focus on recent developments and their implications. It is hoped that knowledge developed from one tissue can be translated to other tissues. Finally, we address challenges in tissue engineering and regenerative medicine that can potentially be overcome via employing strategies for stem cell co-culture use.

Stem cells are the future in tissue engineering and regeneration. In a co-culture, stem cells not only provide a target cell source with multipotent differentiation capacity, but can also act as assisting cells that promote tissue homeostasis, metabolism, growth and repair. Their incorporation into co-culture systems seems to be important in the creation of complex tissues or organs. In this review, critical aspects of stem cell use in co-culture systems are discussed. Direct and indirect co-culture methodologies used in tissue engineering are described, along with various characteristics of cellular interactions in these systems. Direct cell-cell contact, cell-extracellular matrix interaction and signalling via soluble factors are presented. The advantages of stem cell co-culture strategies and their applications in tissue engineering and regenerative medicine are portrayed through specific examples for several tissues, including orthopaedic soft tissues, bone, heart, vasculature, lung, kidney, liver and nerve. A concise review of the progress and the lessons learned are provided, with a focus on recent developments and their implications. It is hoped that knowledge developed from one tissue can be translated to other tissues. Finally, we address challenges in tissue engineering and regenerative medicine that can potentially be overcome via employing strategies for stem cell co-culture use.

Tissue-engineered musculoskeletal soft tissues typically lack the appropriate mechanical robustness of their native counterparts, hindering their clinical applicability. With structure and function being intimately linked, efforts to capture the anatomical shape and matrix organization of native tissues are imperative to engineer functionally robust and anisotropic tissues capable of withstanding the biomechanically complex in vivo joint environment. The present study sought to tailor the use of passive axial compressive loading to drive matrix synthesis and reorganization within self-assembled, shape-specific fibrocartilaginous constructs, with the goal of developing functionally anisotropic neotissues. Specifically, shape-specific fibrocartilaginous neotissues were subjected to 0, 0.01, 0.05, or 0.1 N axial loads early during tissue culture. Results found the 0.1-N load to significantly increase both collagen and glycosaminoglycan synthesis by 27% and 67%, respectively, and to concurrently reorganize the matrix by promoting greater matrix alignment, compaction, and collagen crosslinking compared with all other loading levels. These structural enhancements translated into improved functional properties, with the 0.1-N load significantly increasing both the relaxation modulus and Young's modulus by 96% and 255%, respectively, over controls. Finite element analysis further revealed the 0.1-N uniaxial load to induce multiaxial tensile and compressive strain gradients within the shape-specific neotissues, with maxima of 10.1%, 18.3%, and -21.8% in the XX-, YY-, and ZZ-directions, respectively. This indicates that strains created in different directions in response to a single axis load drove the observed anisotropic functional properties. Together, results of this study suggest that strain thresholds exist within each axis to promote matrix synthesis, alignment, and compaction within the shape-specific neotissues. Tailoring of passive axial loading, thus, presents as a simple, yet effective way to drive in vitro matrix development in shape-specific neotissues toward more closely achieving native structural and functional properties.

Tissue-engineered musculoskeletal soft tissues typically lack the appropriate mechanical robustness of their native counterparts, hindering their clinical applicability. With structure and function being intimately linked, efforts to capture the anatomical shape and matrix organization of native tissues are imperative to engineer functionally robust and anisotropic tissues capable of withstanding the biomechanically complex in vivo joint environment. The present study sought to tailor the use of passive axial compressive loading to drive matrix synthesis and reorganization within self-assembled, shape-specific fibrocartilaginous constructs, with the goal of developing functionally anisotropic neotissues. Specifically, shape-specific fibrocartilaginous neotissues were subjected to 0, 0.01, 0.05, or 0.1 N axial loads early during tissue culture. Results found the 0.1-N load to significantly increase both collagen and glycosaminoglycan synthesis by 27% and 67%, respectively, and to concurrently reorganize the matrix by promoting greater matrix alignment, compaction, and collagen crosslinking compared with all other loading levels. These structural enhancements translated into improved functional properties, with the 0.1-N load significantly increasing both the relaxation modulus and Young's modulus by 96% and 255%, respectively, over controls. Finite element analysis further revealed the 0.1-N uniaxial load to induce multiaxial tensile and compressive strain gradients within the shape-specific neotissues, with maxima of 10.1%, 18.3%, and -21.8% in the XX-, YY-, and ZZ-directions, respectively. This indicates that strains created in different directions in response to a single axis load drove the observed anisotropic functional properties. Together, results of this study suggest that strain thresholds exist within each axis to promote matrix synthesis, alignment, and compaction within the shape-specific neotissues. Tailoring of passive axial loading, thus, presents as a simple, yet effective way to drive in vitro matrix development in shape-specific neotissues toward more closely achieving native structural and functional properties.

The inability to recapitulate native tissue biomechanics, especially tensile properties, hinders progress in regenerative medicine. To address this problem, strategies have focused on enhancing collagen production. However, manipulating collagen cross-links, ubiquitous throughout all tissues and conferring mechanical integrity, has been underinvestigated. A series of studies examined the effects of lysyl oxidase (LOX), the enzyme responsible for the formation of collagen cross-links. Hypoxia-induced endogenous LOX was applied in multiple musculoskeletal tissues (i.e., cartilage, meniscus, tendons, ligaments). Results of these studies showed that both native and engineered tissues are enhanced by invoking a mechanism of hypoxia-induced pyridinoline (PYR) cross-links via intermediaries like LOX. Hypoxia was shown to enhance PYR cross-linking 1.4- to 6.4-fold and, concomitantly, to increase the tensile properties of collagen-rich tissues 1.3- to 2.2-fold. Direct administration of exogenous LOX was applied in native cartilage and neocartilage generated using a scaffold-free, self-assembling process of primary chondrocytes. Exogenous LOX was found to enhance native tissue tensile properties 1.9-fold. LOX concentration- and time-dependent increases in PYR content (∼ 16-fold compared with controls) and tensile properties (approximately fivefold compared with controls) of neocartilage were also detected, resulting in properties on par with native tissue. Finally, in vivo subcutaneous implantation of LOX-treated neocartilage in nude mice promoted further maturation of the neotissue, enhancing tensile and PYR content approximately threefold and 14-fold, respectively, compared with in vitro controls. Collectively, these results provide the first report, to our knowledge, of endogenous (hypoxia-induced) and exogenous LOX applications for promoting collagen cross-linking and improving the tensile properties of a spectrum of native and engineered tissues both in vitro and in vivo.

Patients suffering from damaged or diseased fibrocartilages currently have no effective long-term treatment options. Despite their potential, engineered tissues suffer from inferior biomechanical integrity and an inability to integrate in vivo. The present study identifies a treatment regimen (including the biophysical agent chondroitinase-ABC, the biochemical agent TGF-β1, and the collagen crosslinking agent lysyl oxidase) to prime highly cellularized, scaffold-free neofibrocartilage implants, effecting continued improvement in vivo. We show these agents drive in vitro neofibrocartilage matrix maturation toward synergistically enhanced Young's modulus and ultimate tensile strength values, which were increased 245% and 186%, respectively, over controls. Furthermore, an in vitro fibrocartilage defect model found this treatment regimen to significantly increase the integration tensile properties between treated neofibrocartilage and native tissue. Through translating this technology to an in vivo fibrocartilage defect model, our results indicate, for the first time, that a pre-treatment can prime neofibrocartilage for significantly enhanced integration potential in vivo, with interfacial tensile stiffness and strength increasing by 730% and 745%, respectively, compared to integration values achieved in vitro. Our results suggest that specifically targeting collagen assembly and organization is a powerful means to augment overall neotissue mechanics and integration potential toward improved clinical feasibility.

Patients suffering from damaged or diseased fibrocartilages currently have no effective long-term treatment options. Despite their potential, engineered tissues suffer from inferior biomechanical integrity and an inability to integrate in vivo. The present study identifies a treatment regimen (including the biophysical agent chondroitinase-ABC, the biochemical agent TGF-β1, and the collagen crosslinking agent lysyl oxidase) to prime highly cellularized, scaffold-free neofibrocartilage implants, effecting continued improvement in vivo. We show these agents drive in vitro neofibrocartilage matrix maturation toward synergistically enhanced Young's modulus and ultimate tensile strength values, which were increased 245% and 186%, respectively, over controls. Furthermore, an in vitro fibrocartilage defect model found this treatment regimen to significantly increase the integration tensile properties between treated neofibrocartilage and native tissue. Through translating this technology to an in vivo fibrocartilage defect model, our results indicate, for the first time, that a pre-treatment can prime neofibrocartilage for significantly enhanced integration potential in vivo, with interfacial tensile stiffness and strength increasing by 730% and 745%, respectively, compared to integration values achieved in vitro. Our results suggest that specifically targeting collagen assembly and organization is a powerful means to augment overall neotissue mechanics and integration potential toward improved clinical feasibility.