Combination of a Collagen Scaffold and an Adhesive Hyaluronan-Based Hydrogel for Cartilage Regeneration: A Proof of Concept in an Ovine Model.

OBJECTIVE: Hyaluronic acid-transglutaminase (HA-TG) is an enzymatically crosslinkable adhesive hydrogel with chondrogenic properties demonstrated in vitro and in an ectopic mouse model. In this study, we investigated the feasibility of using HA-TG in a collagen scaffold to treat chondral lesions in an ovine model, to evaluate cartilage regeneration in a mechanically and biologically challenging joint environment, and the influence of the surgical procedure on the repair process. DESIGN: Chondral defects of 6-mm diameter were created in the stifle joint of skeletally mature sheep. In a 3-month study, 6 defects were treated with HA-TG in a collagen scaffold to test the stability and biocompatibility of the defect filling. In a 6-month study, 6 sheep had 12 defects treated with HA-TG and collagen and 2 sheep had 4 untreated defects. Histologically observed quality of repair tissue and adjacent cartilage was semiquantitatively assessed. RESULTS: HA-TG adhered to the native tissue and did not cause any detectable negative reaction in the surrounding tissue. HA-TG in a collagen scaffold supported infiltration and chondrogenic differentiation of mesenchymal cells, which migrated from the subchondral bone through the calcified cartilage layer. Additionally, HA-TG and collagen treatment led to better adjacent cartilage preservation compared with empty defects (P < 0.05). CONCLUSIONS: This study demonstrates that the adhesive HA-TG hydrogel in a collagen scaffold shows good biocompatibility, supports in situ cartilage regeneration and preserves the surrounding cartilage. This proof-of-concept study shows the potential of this approach, which should be further considered in the treatment of cartilage lesions using a single-step procedure.

Tyrosinase-crosslinked, tissue adhesive and biomimetic alginate sulfate hydrogels for cartilage repair.

The native cartilage extracellular matrix (ECM) is enriched in sulfated glycosaminoglycans with important roles in the signaling and phenotype of resident chondrocytes. Recapitulating the key ECM components within engineered tissues through biomimicking strategies has potential to improve the regenerative capacity of encapsulated cells and lead to better clinical outcome. Here, we developed a double-modified, biomimetic and tissue adhesive hydrogel for cartilage engineering. We demonstrated sequential modification of alginate with first sulfate moieties to mimic the high glycosaminoglycan content of native cartilage and then tyramine moieties to allow in situ enzymatic crosslinking with tyrosinase under physiological conditions. Tyrosinase-crosslinked alginate sulfate tyramine (ASTA) hydrogels showed strong adhesion to native cartilage tissue with higher bond strength compared to alginate tyramine (AlgTA). Both ASTA and AlgTA hydrogels supported the viability of encapsulated bovine chondrocytes and induced a strong increase in the expression of chondrogenic genes such as collagen 2, aggrecan and Sox9. Aggrecan and Sox9 gene expression of chondrocytes in ASTA hydrogels were significantly higher than those in AlgTA. Chondrocytes in both ASTA and AlgTA hydrogels showed potent deposition of cartilage matrix components collagen 2 and aggrecan after 3 weeks of culture whereas a decreased collagen 1 deposition was observed in the sulfated hydrogels. ASTA and AlgTA hydrogels with encapsulated human chondrocytes showed in vivo stability as well as cartilage matrix deposition upon subcutaneous implantation into mice for 4 weeks. Our data is the first demonstration of a double-modified alginate with sulfation and tyramination that allows in situ enzymatic crosslinking, strong adhesion to native cartilage and chondrogenic re-differentiation.

Nanocomposite bioink exploits dynamic covalent bonds between nanoparticles and polysaccharides for precision bioprinting.

The field of bioprinting has made significant recent progress towards engineering tissues with increasing complexity and functionality. It remains challenging, however, to develop bioinks with optimal biocompatibility and good printing fidelity. Here, we demonstrate enhanced printability of a polymer-based bioink based on dynamic covalent linkages between nanoparticles (NPs) and polymers, which retains good biocompatibility. Amine-presenting silica NPs (ca. 45 nm) were added to a polymeric ink containing oxidized alginate (OxA). The formation of reversible imine bonds between amines on the NPs and aldehydes of OxA lead to significantly improved rheological properties and high printing fidelity. In particular, the yield stress increased with increasing amounts of NPs (14.5 Pa without NPs, 79 Pa with 2 wt% NPs). In addition, the presence of dynamic covalent linkages in the gel provided improved mechanical stability over 7 d compared to ionically crosslinked gels. The nanocomposite ink retained high printability and mechanical strength, resulting in generation of centimeter-scale porous constructs and an ear structure with overhangs and high structural fidelity. Furthermore, the nanocomposite ink supported both in vitro and in vivo maturation of bioprinted gels containing chondrocytes. This approach based on simple oxidation can be applied to any polysaccharide, thus the widely applicability of the method is expected to advance the field towards the goal of precision bioprinting.

Is There a Scientific Rationale for the Refixation of Delaminated Chondral Flaps in Femoroacetabular Impingement? A Laboratory Study.

BACKGROUND: Debonding of the acetabular cartilage is a characteristic type of hip damage found in cam-type femoroacetabular impingement (FAI), which remains a treatment challenge. In addition to resection, refixation of these flaps using fibrin sealants has been recently suggested. However, there is only limited evidence available that the proposed refixation method results in sufficient viable cartilage formation to ensure long-term flap grafting and restored tissue function. QUESTIONS/PURPOSES: To determine the flap tissue characteristics that would justify refixation of delaminated chondral flaps with a fibrin sealant, we characterized (1) the extracellular matrix (ECM) of chondral flaps in terms of chondrocyte viability and distribution of ECM components and (2) the chondrogenic potential of resident cells to migrate into fibrin and produce a cartilaginous matrix. METHODS: Ten acetabular chondral flaps and three non-delaminated control cartilage samples were resected during surgery. Chondrocyte viability was quantified using a live-dead assay. To assess the ECM, histological staining of glycosaminoglycans, collagen II, and collagen I allowed the qualitative study of their distribution. The ability of chondrocytes to migrate out of the ECM was tested by encapsulating minced flap cartilage in fibrin gels and semi-quantitatively assessing the projected area of the gel covered with migrating cells. The potential of chondrocytes to produce a cartilaginous matrix was studied with a pellet assay, a standard three-dimensional culture system to test chondrogenesis. Positive controls were pellets of knee chondrocytes of age-matched donors, which we found in a previous study to have a good capacity to produce cartilage matrix. Statistical significance of controlled quantitative assays was determined by the Student's t-test with Welch's correction. RESULTS: The proportion of viable chondrocytes in flaps was lower than in nondelaminated cartilage (50% ± 19% versus 76 ± 6%; p = 0.02). Histology showed a disrupted ECM in flaps compared with nondelaminated controls, with the presence of fibrillation, a loss of glycosaminoglycan at the delaminated edge, collagen II throughout the whole thickness of the flap, and some collagen I-positive area in two samples. The resident chondrocytes migrated out of this disrupted ECM in all tested samples. However in pellet culture, cells isolated from the flaps showed a qualitatively lower chondrogenic potential compared with positive controls, with a clearly inhomogeneous cell and matrix distribution and an overall smaller projected area (0.4 versus 0.7 mm; p = 0.038). CONCLUSION: Despite the presence of viable chondrocytes with migration potential, the cells resided in a structurally altered ECM and had limited capacity to deposit ECM, leading us to question their capacity to produce sufficient ECM within the fibrin sealant for stable long-term attachment of such flaps. CLINICAL RELEVANCE: The characterization of delaminated cartilage in cam FAI patients suggests that the refixation strategy might be adversely influenced by the low level of ECM produced by the residing cells.

Microencapsulation improves chondrogenesis in vitro and cartilaginous matrix stability in vivo compared to bulk encapsulation.

The encapsulation of cells into microgels is attractive for applications in tissue regeneration. While cells are protected against shear stress during injection, the assembly of microgels after injection into a tissue defect also forms a macroporous scaffold that allows effective nutrient transport throughout the construct. However, in most of current strategies that form microgel-based macroporous scaffold or higher-order structures, cells are seeded during or post the assembly process and not microencapsulated in situ. The objective of this study is to investigate the chondrogenic phenotype of microencapsulated fetal chondrocytes in a biocompatible, assembled microgel system vs. bulk gels and to test the stability of the constructs in vivo. Here, we demonstrate that cell microencapsulation leads to increased expression of cartilage-specific genes in a TGF-ß1-dependent manner. This correlates, as shown by histological staining, with the ability of microencapsulated cells to deposit cartilaginous matrix after migrating to the surface of the microgels, while keeping a macroscopic granular morphology. Implantation of precultured scaffolds in a subcutaneous mouse model results in vessel infiltration in bulk gels but not in assembled microgels, suggesting a higher stability of the matrix produced by the cells in the assembled microgel constructs. The cells are able to remodel the microgels as demonstrated by the gradual disappearance of the granular structure in vivo. The biocompatible microencapsulation and microgel assembly system presented in this article therefore hold great promise as an injectable system for cartilage repair.

Chondrocytes From Device-Minced Articular Cartilage Show Potent Outgrowth Into Fibrin and Collagen Hydrogels.

BACKGROUND: Transplantation of autologous minced cartilage is an established procedure to repair chondral lesions. It relies on the migration of chondrocytes out of cartilage particles into a biomaterial. So far, there is no efficient way to finely mince cartilage. No consensus exists on the nature of the biomaterial to be used to promote chondrocyte migration. PURPOSE/HYPOTHESIS: This study aimed to investigate the potential clinical use of a custom-made mincing device as well as a possible alternative biomaterial to fibrin glue. The device was tested for its effect on chondrocyte viability and on subsequent chondrocyte migration into either a fibrin or a collagen gel. We hypothesized that device mincing would allow finer cutting and consequently more cell migration and that the gelation mechanism of the collagen biomaterial, which uses the clotting of platelet-rich plasma, would enhance matrix production by outgrown chondrocytes. STUDY DESIGN: Controlled laboratory study. METHODS: Cartilage from 12 patients undergoing knee arthroplasty was taken from the femoral condyles and subsequently either hand minced or device minced. The viability and the degree of outgrowth were quantified with live/dead assay on the generated cartilage particles and on the gels in which these particles were embedded, respectively. Matrix deposition in the biomaterials by the outgrown cells was investigated with histology. RESULTS: The device allowed rapid mincing of the cartilage and produced significantly smaller pieces than hand mincing. The initial chondrocyte viability in cartilage particles dropped by 25% with device mincing as compared with no mincing. However, the viability in hand-minced, device-minced, and unminced samples was no longer different after 7 and 28 days in culture. Outgrowth scores were similar among the 3 groups. Fibrin and collagen biomaterials equally supported chondrocyte outgrowth and survival, but neither promoted matrix deposition after in vitro culture. CONCLUSION: The outgrowth potential, the viability after 28 days in culture, and the matrix deposition were not different between the mincing techniques and the tested biomaterials, yet device mincing is faster and results in significantly smaller cartilage particles. CLINICAL RELEVANCE: Device mincing could become the standard method to mince cartilage for second-generation cartilage repair techniques.

An injectable heparin-conjugated hyaluronan scaffold for local delivery of transforming growth factor ß1 promotes successful chondrogenesis.

Cartilage lacks basic repair mechanisms and thus surgical interventions are necessary to treat lesions. Minimally-invasive arthroscopic procedures require the development of injectable biomaterials to support chondrogenesis of implanted cells. However, most cartilage tissue engineering approaches rely on pre-culture of scaffolds in media containing growth factors (GFs) such as transforming growth factor (TGF)-ß1, which are crucial for cartilage formation and homeostasis. GFs media-supplementation is incompatible with injectable approaches and has led to a knowledge gap about optimal dose of GFs and release profiles needed to achieve chondrogenesis. This study aims to determine the optimal loading and release kinetics of TGF-ß1 bound to an engineered GAG hydrogel to promote optimal cartilaginous matrix production in absence of TGF-ß1 media-supplementation. We show that heparin, a GAG known to bind a wide range of GFs, covalently conjugated to a hyaluronan hydrogel, leads to a sustained release of TGF-ß1. Using this heparin-conjugated hyaluronan hydrogel, 0.25 to 50 ng TGF-ß1 per scaffold was loaded and cell viability, proliferation and cartilaginous matrix deposition of the encapsulated chondroprogenitor cells were measured. Excellent chondrogenesis was found when 5 ng TGF-ß1 per scaffold and higher were used. We also demonstrate the necessity of a sustained release of TGF-ß1, as no matrix deposition is observed upon a burst release. In conclusion, our biomaterial loaded with an optimal initial dose of 5 ng/scaffold TGF-ß1 is a promising injectable material for cartilage repair, with potentially increased safety due to the low, locally administered GF dose. STATEMENT OF SIGNIFICANCE: Cartilage cell-based products are dependent on exogenous growth factor supplementation in order for proper tissue maturation. However, for a one-step repair of defects without need for expensive tissue maturation, an injectable, growth factor loaded formulation is required. Here we show development of an injectable hyaluronan hydrogel, which achieves a sustained release of TGF-ß1 due to covalent conjugation of heparin. These grafts matured into cartilaginous tissue in the absence of growth factor supplementation. Additionally, this system allowed us to screen TGF-ß1 concentrations to determine the mimimum amount of growth factor required for chondrogenesis. This study represents a critical step towards development of a minimally-invasive, arthroscopic treatment for cartilage lesions.

Characterization of polydactyly chondrocytes and their use in cartilage engineering.

Treating cartilage injuries and degenerations represents an open surgical challenge. The recent advances in cell therapies have raised the need for a potent off-the-shelf cell source. Intra-articular injections of TGF-ß transduced polydactyly chondrocytes have been proposed as a chronic osteoarthritis treatment but despite promising results, the use of gene therapy still raises safety concerns. In this study, we characterized infant, polydactyly chondrocytes during in vitro expansion and chondrogenic re-differentiation. Polydactyly chondrocytes have a steady proliferative rate and re-differentiate in 3D pellet culture after up to five passages. Additionally, we demonstrated that polydactyly chondrocytes produce cartilage-like matrix in a hyaluronan-based hydrogel, namely transglutaminase cross-linked hyaluronic acid (HA-TG). We utilized the versatility of TG cross-linking to augment the hydrogels with heparin moieties. The heparin chains allowed us to load the scaffolds with TGF-ß1, which induced cartilage-like matrix deposition both in vitro and in vivo in a subcutaneous mouse model. This strategy introduces the possibility to use infant, polydactyly chondrocytes for the clinical treatment of joint diseases.

Cartilage tissue formation through assembly of microgels containing mesenchymal stem cells.

Current clinical approaches to treat articular cartilage degeneration provide only a limited ability to regenerate tissue with long-term durability and functionality. In this application, injectable bulk hydrogels and microgels containing stem cells can provide a suitable environment for tissue regeneration. However insufficient cell-cell interactions, low differentiation efficiency and poor tissue adhesion hinder the formation of high-quality hyaline type cartilage. Here, we have designed a higher order tissue-like structure using injectable cell-laden microgels as the building blocks to achieve human bone marrow-derived mesenchymal stem cell (hBMSC) long-term maintenance and chondrogenesis. We have demonstrated that a 4-arm poly(ethylene glycol)-N-hydroxysuccinimide (NHS) crosslinker induces covalent bonding between the microgel building blocks as well as the surrounding tissue mimic. The crosslinking process assembles the microgels into a 3D construct and preserves the viability and cellular functions of the encapsulated hBMSCs. This assembled microgel construct encourages upregulation of chondrogenic markers in both gene and glycosaminoglycan (GAG) expression levels. In addition, the regenerated tissue in the assembled microgels stained positively with Alcian blue and Safranin O exhibiting unique hyaline-like cartilage features. Furthermore, the immunostaining showed a favourable distribution and significantly higher content of type II collagen in the assembled microgels when compared to both the bulk hydrogel and pellet cultures. Collectively, this tissue adhesive hBMSC-laden microgel construct provides potential clinical opportunities for articular cartilage repair and other applications in regenerative medicine. STATEMENT OF SIGNIFICANCE: A reliable approach to reconstruct durable and fully functional articular cartilage tissue is required for effective clinical therapies. Here, injectable hydrogels together with cell-based therapies offer new treatment strategies in cartilage repair. For effective cartilage regeneration, the injectable hydrogel system needs to be bonded to the surrounding tissue and at the same time needs to be sufficiently stable for prolonged chondrogenesis. In this work, we utilised injectable hBMSC-laden microgels as the building blocks to create an assembled construct via N-hydroxysuccinimide-amine coupling. This crosslinking process also allows for rapid bonding between the assembled microgels and a surrounding tissue mimic. The resultant assembled microgel-construct provides both a physically stable and biologically dynamic environment for hBMSC chondrogenesis, leading to the production of a mature hyaline type cartilage structure.