Cartilage Tissue Engineering Approaches Research Articles

Glycosaminoglycan-Mediated Interactions in Articular, Auricular, Meniscal, and Nasal Cartilage.

Glycosaminoglycans (GAGs) are ubiquitous components in the cartilage extracellular matrix (ECM). Ultrastructural arrangement of ECM and GAG-mediated interactions with collagen are known to govern the mechanics in articular cartilage, but these interactions are less clear in other cartilage types. Therefore, this article reviews the current literature on ultrastructure of articular, auricular, meniscal, and nasal septal cartilage, seeking insight into GAG-mediated interactions influencing mechanics. Ultrastructural features of these cartilages are discussed to highlight differences between them. GAG-mediated interactions are reviewed under two categories: interactions with chondrocytes and interactions with other fibrillar macromolecules of the ECM. Moreover, efforts to replicate GAG-mediated interactions to improve mechanical integrity of tissue-engineered cartilage constructs are discussed. In conclusion, studies exploring cartilage specific GAGs are poorly represented in the literature, and the ultrastructure of nasal septal and auricular cartilage is less studied compared with articular and meniscal cartilages. Understanding the contribution of GAGs in cartilage mechanics at the ultrastructural level and translating that knowledge to engineered cartilage will facilitate improvement of cartilage tissue engineering approaches.

Applied Compressive Strain Governs Hyaline-like Cartilage versus Fibrocartilage-like ECM Produced within Hydrogel Constructs.

The goal of cartilage tissue engineering (CTE) is to regenerate new hyaline cartilage in joints and treat osteoarthritis (OA) using cell-impregnated hydrogel constructs. However, the production of an extracellular matrix (ECM) made of fibrocartilage is a potential outcome within hydrogel constructs when in vivo. Unfortunately, this fibrocartilage ECM has inferior biological and mechanical properties when compared to native hyaline cartilage. It was hypothesized that compressive forces stimulate fibrocartilage development by increasing production of collagen type 1 (Col1), an ECM protein found in fibrocartilage. To test the hypothesis, 3-dimensional (3D)-bioprinted hydrogel constructs were fabricated from alginate hydrogel impregnated with ATDC5 cells (a chondrogenic cell line). A bioreactor was used to simulate different in vivo joint movements by varying the magnitude of compressive strains and compare them with a control group that was not loaded. Chondrogenic differentiation of the cells in loaded and unloaded conditions was confirmed by deposition of cartilage specific molecules including glycosaminoglycans (GAGs) and collagen type 2 (Col2). By performing biochemical assays, the production of GAGs and total collagen was also confirmed, and their contents were quantitated in unloaded and loaded conditions. Furthermore, Col1 vs. Col2 depositions were assessed at different compressive strains, and hyaline-like cartilage vs. fibrocartilage-like ECM production was analyzed to investigate how applied compressive strain affects the type of cartilage formed. These assessments showed that fibrocartilage-like ECM production tended to reduce with increasing compressive strain, though its production peaked at a higher compressive strain. According to these results, the magnitude of applied compressive strain governs the production of hyaline-like cartilage vs. fibrocartilage-like ECM and a high compressive strain stimulates fibrocartilage-like ECM formation rather than hyaline cartilage, which needs to be addressed by CTE approaches.

Progress of Platelet Derivatives for Cartilage Tissue Engineering.

Articular cartilage has limited self-regeneration ability for lacking of blood vessels, nerves, and lymph that makes it a great challenge to repair defects of the tissue and restore motor functions of the injured or aging population. Platelet derivatives, such as platelet-rich plasma, have been proved effective, safe, and economical in musculoskeletal diseases for their autologous origin and rich in growth factors. The combination of platelet derivatives with biomaterials provides both mechanical support and localized sustained release of bioactive molecules in cartilage tissue engineering and low-cost efficient approaches of potential treatment. In this review, we first provide an overview of platelet derivatives and their application in clinical and experimental therapies, and then we further discuss the techniques of the addition of platelet derivatives and their influences on scaffold properties. Advances in cartilage tissue engineering with platelet derivatives as signal factors and structural components are also introduced before prospects and concerns in this research field. In short, platelet derivatives have broad application prospects as an economical and effective enhancement for tissue engineering–based articular cartilage repair.

Cartilage Tissue Engineering Approaches Need to Assess Fibrocartilage When Hydrogel Constructs Are Mechanically Loaded.

Chondrocytes that are impregnated within hydrogel constructs sense applied mechanical force and can respond by expressing collagens, which are deposited into the extracellular matrix (ECM). The intention of most cartilage tissue engineering is to form hyaline cartilage, but if mechanical stimulation pushes the ratio of collagen type I (Col1) to collagen type II (Col2) in the ECM too high, then fibrocartilage can form instead. With a focus on Col1 and Col2 expression, the first part of this article reviews the latest studies on hyaline cartilage regeneration within hydrogel constructs that are subjected to compression forces (one of the major types of the forces within joints) in vitro. Since the mechanical loading conditions involving compression and other forces in joints are difficult to reproduce in vitro, implantation of hydrogel constructs in vivo is also reviewed, again with a focus on Col1 and Col2 production within the newly formed cartilage. Furthermore, mechanotransduction pathways that may be related to the expression of Col1 and Col2 within chondrocytes are reviewed and examined. Also, two recently-emerged, novel approaches of load-shielding and synchrotron radiation (SR)–based imaging techniques are discussed and highlighted for future applications to the regeneration of hyaline cartilage. Going forward, all cartilage tissue engineering experiments should assess thoroughly whether fibrocartilage or hyaline cartilage is formed.

Targeted Activation of G-Protein Coupled Receptor-Mediated Ca2+ Signaling Drives Enhanced Cartilage-Like Matrix Formation.

Intracellular calcium ([Ca2+]i) signaling is a critical regulator of chondrogenesis, chondrocyte differentiation, and cartilage development. Calcium (Ca2+) signaling is known to direct processes that govern chondrocyte gene expression, protein synthesis, cytoskeletal remodeling, and cell fate. Control of chondrocyte/chondroprogenitor Ca2+ signaling has been attempted through mechanical and/or pharmacological activation of endogenous Ca2+ signaling transducers; however, such approaches can lack specificity and/or precision regarding Ca2+ activation mechanisms. Synthetic signaling platforms permitting precise and selective Ca2+ signal transduction can improve dissection of the roles that [Ca2+]i signaling plays in chondrocyte behavior. One such platform is the chemogenetic DREADD (designer receptor exclusively activated by designer drugs) hM3Dq, which activates [Ca2+]i signaling via the Gαq-PLCβ-IP3-ER pathway upon clozapine N-oxide (CNO) administration. We previously demonstrated hM3Dq's ability to precisely and synthetically initiate robust [Ca2+]i transients and oscillatory [Ca2+]i signaling in chondrocyte-like ATDC5 cells. Here, we investigate the effects that long-term CNO stimulatory culture have on hM3Dq [Ca2+]i signaling dynamics, proliferation, and protein deposition in 2D ATDC5 cultures. Long-term culturing under repeated CNO stimulation modified the temporal dynamics of hM3Dq [Ca2+]i signaling, increased cell proliferation, and enhanced matrix production in a CNO dose- and frequency-dependent manner, and triggered the formation of cell condensations that developed aligned, anisotropic neotissue structures rich in cartilaginous proteoglycans and collagens, all in the absence of differentiation inducers. This study demonstrated Gαq-G-protein coupled receptor (GPCR)-mediated [Ca2+]i signaling involvement in chondroprogenitor proliferation and cartilage-like matrix production, and it established hM3Dq as a powerful tool for elucidating the role of GPCR-mediated Ca2+ signaling in chondrogenesis and chondrocyte differentiation. Impact statement Targeted activation of intracellular calcium signaling has gained attention as a cartilage tissue engineering adjuvant approach. In the present study, we demonstrated that activation of hM3Dq, an engineered chemogenetic activator of the Gαq-pathway and IP3-mediated intracellular calcium signaling, drives accelerated development of mesenchyme-like cell condensations and cartilaginous neotissue formation in chondrocyte-like cell cultures in vitro and does so without the requirement of differentiation factors/inducers. These outcomes highlight the potential of targeted/synthetic Gαq-pathway activation, specifically using novel chemogenetic approaches, to enhance the study of chondrocyte physiology and improve cartilage tissue engineering approaches.

Bioprinting of a Zonal-Specific Cell Density Scaffold: A Biomimetic Approach for Cartilage Tissue Engineering

The treatment of articular cartilage defects remains a significant clinical challenge. This is partially due to current tissue engineering strategies failing to recapitulate native organization. Articular cartilage is a graded tissue with three layers exhibiting different cell densities: the superficial zone having the highest density and the deep zone having the lowest density. However, the introduction of cell gradients for cartilage tissue engineering, which could promote a more biomimetic environment, has not been widely explored. Here, we aimed to bioprint a scaffold with different zonal cell densities to mimic the organization of articular cartilage. The scaffold was bioprinted using an alginate-based bioink containing human articular chondrocytes. The scaffold design included three cell densities, one per zone: 20 × 106 (superficial), 10 × 106 (middle), and 5 × 106 (deep) cells/mL. The scaffold was cultured in a chondrogenic medium for 25 days and analyzed by live/dead assay and histology. The live/dead analysis showed the ability to generate a zonal cell density with high viability. Histological analysis revealed a smooth transition between the zones in terms of cell distribution and a higher sulphated glycosaminoglycan deposition in the highest cell density zone. These findings pave the way toward bioprinting complex zonal cartilage scaffolds as single units, thereby advancing the translation of cartilage tissue engineering into clinical practice.

Platelet-rich plasma-hyaluronic acid/chondrotin sulfate/carboxymethyl chitosan hydrogel for cartilage regeneration.

In this study, the chondrogenic potential of hyaluronic acid/chondrotin sulfate/carboxymethyl chitosan hydrogels with adipose-derived mesenchymal stem cells (ADMSCs) was evaluated. Here, hyaluronic acid, chondrotin sulfate, and carboxymethyl chitosan were used as the substrate for cartilage tissue engineering in which the hydrogel is formed due to electrostatic and hydrogen bonds through mixing the polymers. Because of the instability of this hydrogel in the biological environment, 1-ethyl-3-(3-dimethylaminopropyl-carbodiimide hydrochloride/N-hydroxy-succinimide was used as a crosslinker to increase the hydrogel stability. The hydrogels showed reasonable stability due to the combined effect of self-crosslinking and chemical crosslinking. The cells were treated with the prepared hydrogel samples for 14 and 21 days in nondifferentiation medium for evaluation of the cellular behavior of ADMSCs. Gene expression evaluation was performed, and expression of specific genes involved in differentiation was shown in the crosslinked hydrogel with platelet-rich plasma (PRP) (H-EN-P) had increased the gene expression levels. Quantification of immunofluorescence intensity indicated the high level of expression of SOX9 in H-EN-P hydrogel. Based on the results, we confirmed that the presence of PRP and the similarity of the hydrogel constituents to the cartilage extracellular matrix could have positive effects on the differentiation of the cells, which is favorable for cartilage tissue engineering approaches.

Pre-Culture of Human Mesenchymal Stromal Cells in Spheroids Reduces Hypertrophic Differentiation <i> in vitro</i> and Facilitates Chondrogenesis at a Low Total Cell Count Upon Embedding in Biomaterials

Material assisted cartilage tissue engineering approaches have limited application in cartilage treatment due to hypertrophic and fibrocartilaginous tissue formation and the high cell count needed. The aim of this study was to investigate the potential of human mesenchymal stromal cell (hMSC) spheroids embedded in biomaterials to study the effect of biomaterial composition on cell differentiation. hMSCs were pre-cultured in spheroids (250 cells/spheroid) for three days in chondrogenic differentiation media. Spheroids were embedded in tyramine-modified hyaluronic acid (THA) and collagen type I (Col) composite hydrogels at four combinations of THA (12.5 vs 16.7 mg/ml) and Col (2.5 vs 1.7 mg/ml) content at a cell density of 5 Mio cells/ml (1’000 spheroids/sample). Macro-pellets derived from hMSCs (250’000 cells, MP) or hMSC spheroids (250’000 cells/1’000 spheroids, SP) served as controls. hMSCs differentiation (day 21) was analyzed using quantification of glycosaminoglycans (GAGs), gene expression analysis and (immuno-) histological stainings. Embedding of hMSC spheroids in THA-Col biomaterials induced chondrogenic differentiation marked by upregulation of aggrecan (ACAN) and COL2A1, and production of GAGs. A lower THA content led to a more pronounced chondrogenic phenotype compared to a higher THA content. Col content had no significant influence on hMSC chondrogenesis. Pellet cultures (MP, SP) showed an upregulation in chondrogenic-associated genes and production of GAGs with less upregulation of hypertrophic-associated genes in MP culture compared to SP group.This study indicated a positive benefit of pre-culturing hMSCs in spheroids with respect to hypertrophic gene expression compared to the gold standard pellet culture without compromising chondrogenic matrix production. Funding: This work is part of the osteochondral defect collaborative research program supported by the AO Foundation. F.S. received the AO Foundation Fellowship in Health Sciences and Technology from ETH Zurich Foundation. Declaration of Interests: The authors have no competing of interests. Ethics Approval Statement: Full ethical approval (Inselspital Bern - Bern Req-2016-00141, University hospital Freiburg - EKFreiburg: 135/14).

Cartilage Extracellular Matrix Scaffold With Kartogenin-Encapsulated PLGA Microspheres for Cartilage Regeneration.

Repair of articular cartilage defects is a challenging aspect of clinical treatment. Kartogenin (KGN), a small molecular compound, can induce the differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) into chondrocytes. Here, we constructed a scaffold based on chondrocyte extracellular matrix (CECM) and poly(lactic-co-glycolic acid) (PLGA) microspheres (MP), which can slowly release KGN, thus enhancing its efficiency. Cell adhesion, live/dead staining, and CCK-8 results indicated that the PLGA(KGN)/CECM scaffold exhibited good biocompatibility. Histological staining and quantitative analysis demonstrated the ability of the PLGA(KGN)/CECM composite scaffold to promote the differentiation of BMSCs. Macroscopic observations, histological tests, and specific marker analysis showed that the regenerated tissues possessed characteristics similar to those of normal hyaline cartilage in a rabbit model. Use of the PLGA(KGN)/CECM scaffold may mimic the regenerative microenvironment, thereby promoting chondrogenic differentiation of BMSCs in vitro and in vivo. Therefore, this innovative composite scaffold may represent a promising approach for acellular cartilage tissue engineering.

Graphene Oxide Reinforcement Enhances the Piezoelectric and Mechanical Properties of Poly(3-hydroxybutyrate-co-3-hydroxy valerate)-Based Nanofibrous Scaffolds for Improved Proliferation of Chondrocytes and ECM Production.

The discovery of piezoelectricity in natural cartilage has inspired the development of piezoelectric biomaterials for its repair and regeneration using tissue engineering approaches. In the present work, piezoelectric scaffolds composed of poly(3-hydroxybutyrate-co-3-hydroxy valerate) (PB) and graphene oxide (GO) have been successfully fabricated by the electrospinning technology. The fabricated scaffolds were examined for their morphological, physical, chemical, piezoelectric, and biological characterizations. The fiber diameter was found to be in the range of 600-800 nm appropriate for chondrogenic growth. Reinforcement of 1.5% GO enhanced the tensile strength of PB to 2.08 ± 0.33 MPa compared to PB alone (0.59 ± 0.12). Reinforcement of GO significantly enhances the piezoelectric coefficient (d33), and for 0.5, 1, and 1.5% GO in PB, it was found to be 0.12 ± 0.015, 0.57 ± 0.19, and 0.94 ± 0.03 pC/N, respectively, and corresponding voltages of 11.84 ± 1.4, 54.69 ± 18.29, and 100.2 ± 3.2 mV, respectively, were generated. The biological activity of the smart piezo scaffolds was also evaluated on freshly isolated goat chondrocytes. The GO-reinforced scaffold showed higher cell proliferation and cell adhesion as confirmed by alamarBlue assay and field emission scanning electron microscopy imaging. The GO-reinforced scaffold has demonstrated significantly higher extracellular matrix production compared to PB as confirmed by histochemistry and real-time polymerase chain reaction. Hence, the GO-based piezoelectric PB electrospun scaffold can be a better alternative for cell-free and growth factor-free approach for cartilage tissue engineering.

Autologous stem cell-derived chondrocyte implantation with bio-targeted microspheres for the treatment of osteochondral defects

BackgroundChondral injury is a common problem around the world. Currently, there are several treatment strategies for these types of injuries. The possible complications and problems associated with conventional techniques lead us to investigate a minimally invasive and biotechnological alternative treatment. Combining tissue-engineering and microencapsulation technologies provide new direction for the development of biotechnological solutions. The aim of this study is to develop a minimal invasive tissue-engineering approach, using bio-targeted microspheres including autologous cells, for the treatment of the cartilage lesions.MethodIn this study, a total of 28 sheeps of Akkaraman breed were randomly assigned to one of the following groups: control (group 1), microfracture (group 2), scaffold (group 3), and microsphere (group 4). Microspheres and scaffold group animals underwent adipose tissue collection prior to the treatment surgery. Mesenchymal cells collected from adipose tissue were differentiated into chondrocytes and encapsulated with scaffolds and microspheres. Osteochondral damage was conducted in the right knee joint of the sheep to create an animal model and all animals treated according to study groups.ResultsBoth macroscopic and radiologic examination showed that groups 3 and 4 have resulted better compared to the control and microfracture groups. Moreover, histologic assessments indicate hyaline-like cartilage formations in groups 3 and 4.ConclusionIn conclusion, we believe that the bio-targeted microspheres can be a more effective, easier, and safer approach for cartilage tissue engineering compared to previous alternatives.

An injectable heparin-conjugated hyaluronan scaffold for local delivery of transforming growth factor β1 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)-β1, 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-β1 bound to an engineered GAG hydrogel to promote optimal cartilaginous matrix production in absence of TGF-β1 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-β1. Using this heparin-conjugated hyaluronan hydrogel, 0.25 to 50 ng TGF-β1 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-β1 per scaffold and higher were used. We also demonstrate the necessity of a sustained release of TGF-β1, 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-β1 is a promising injectable material for cartilage repair, with potentially increased safety due to the low, locally administered GF dose. Statement of SignificanceCartilage 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-β1 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-β1 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.

Collagen Scaffolds in Cartilage Tissue Engineering and Relevant Approaches for Future Development.

Cartilage tissue engineering (CTE) aims to obtain a structure mimicking native cartilage tissue through the combination of relevant cells, three-dimensional scaffolds, and extraneous signals. Implantation of 'matured' constructs is thus expected to provide solution for treating large injury of articular cartilage. Type I collagen is widely used as scaffolds for CTE products undergoing clinical trial, owing to its ubiquitous biocompatibility and vast clinical approval. However, the long-term performance of pure type I collagen scaffolds would suffer from its limited chondrogenic capacity and inferior mechanical properties. This paper aims to provide insights necessary for advancing type I collagen scaffolds in the CTE applications. Initially, the interactions of type I/II collagen with CTE-relevant cells [i.e., articular chondrocytes (ACs) and mesenchymal stem cells (MSCs)] are discussed. Next, the physical features and chemical composition of the scaffolds crucial to support chondrogenic activities of AC and MSC are highlighted. Attempts to optimize the collagen scaffolds by blending with natural/synthetic polymers are described. Hybrid strategy in which collagen and structural polymers are combined in non-blending manner is detailed. Type I collagen is sufficient to support cellular activities of ACs and MSCs; however it shows limited chondrogenic performance than type II collagen. Nonetheless, type I collagen is the clinically feasible option since type II collagen shows arthritogenic potency. Physical features of scaffolds such as internal structure, pore size, stiffness, etc. are shown to be crucial in influencing the differentiation fate and secreting extracellular matrixes from ACs and MSCs. Collagen can be blended with native or synthetic polymer to improve the mechanical and bioactivities of final composites. However, the versatility of blending strategy is limited due to denaturation of type I collagen at harsh processing condition. Hybrid strategy is successful in maximizing bioactivity of collagen scaffolds and mechanical robustness of structural polymer. Considering the previous improvements of physical and compositional properties of collagen scaffolds and recent manufacturing developments of structural polymer, it is concluded that hybrid strategy is a promising approach to advance further collagen-based scaffolds in CTE.

Co-culture of adipose-derived stem cells and chondrocytes on three-dimensionally printed bioscaffolds for craniofacial cartilage engineering.

Reconstruction of craniofacial cartilagenous defects are among the most challenging surgical procedures in facial plastic surgery. Bioengineered craniofacial cartilage holds immense potential to surpass current reconstructive options, but limitations to clinical translation exist. We endeavored to determine the viability of utilizing adipose-derived stem cell-chondrocyte co-culture and three-dimensional (3D) printing to produce 3D bioscaffolds for cartilage tissue engineering. We describe a feasibility study revealing a novel approach for cartilage tissue engineering with in vitro and in vivo animal data. Porcine adipose-derived stem cells and chondrocytes were isolated and co-seeded at 1:1, 2:1, 5:1, 10:1, and 0:1 experimental ratios in a hyaluronic acid/collagen hydrogel in the pores of 3D-printed polycaprolactone scaffolds to form 3D bioscaffolds for cartilage tissue engineering. Bioscaffolds were cultured in vitro without growth factors for 4 weeks and then implanted into the subcutaneous tissue of athymic rats for an additional 4 weeks before sacrifice. Bioscaffolds were subjected to histologic, immunohistochemical, and biochemical analysis. Successful production of cartilage was achieved using a co-culture model of adipose-derived stem cells and chondrocytes without the use of exogenous growth factors. Histology demonstrated cartilage growth for all experimental ratios at the post-in vivo time point confirmed with type II collagen immunohistochemistry. There was no difference in sulfated-glycosaminoglycan production between experimental groups. Tissue-engineered cartilage was successfully produced on 3D-printed bioresorbable scaffolds using an adipose-derived stem cell and chondrocyte co-culture technique. This potentiates co-culture as a solution for several key barriers to a clinically translatable cartilage tissue engineering process. NA. Laryngoscope, 128:E251-E257, 2018.

Chondroinductive factor-free chondrogenic differentiation of human mesenchymal stem cells in graphene oxide-incorporated hydrogels.

Graphene-based nanomaterials have been applied as biomaterials to enhance stem cell adhesion, growth and differentiation by serving as nanocarriers for growth factors or other small molecules. However, the direct effect of graphene oxide (GO) itself on stem cells, in the absence of exogenous differentiation inductive factors, has not been tested. In this study, we loaded GO nanosheets and human bone marrow-derived mesenchymal stem cells (hBMSC) into a photopolymerizable poly-d,l-lactic acid/polyethylene glycol (PDLLA) hydrogel, a robust chondrosupportive scaffold recently developed in our laboratory, and assessed hBMSC differentiation along the chondrogenic lineage without supplemental chondroinductive factors. We first examined the effect of GO incorporation on the mechanical properties of constructs, and observed that the GO-containing constructs (GO/PDLLA) exhibited enhanced compressive modulus in a GO concentration dependent manner. hBMSCs cultured in GO/PDLLA maintained high cell viability (>95%), indicating minimal cytotoxicity of GO. Importantly, compared to those encapsulated in PDLLA hydrogel, hBMSCs within GO/PDLLA showed significantly higher level of gene expression of the cartilage matrix genes, aggrecan and collagen type II, and produced more cartilage matrix. In addition, the pro-chondrogenesis effect of GO increased with increasing GO concentration. Immunohistochemical results suggested that GO-enhanced hBMSC chondrogenesis was correlated with enriched sequestration of insulin, a necessary supplement known to have pro-chondrogenesis effects on hBMSC. Taken together, these findings demonstrate the utility of using GO to improve the mechanical properties and chondrogenic differentiation state of MSC-laden, engineered hydrogel constructs, without the use of exogenous growth factors, thus representing a potentially promising, biologics-free approach for cartilage tissue engineering.

Chondrocyte burst promotes space for mineral expansion.

Analysis of tissue development from multidisciplinary approaches can result in more integrative biological findings, and can eventually allow the development of more effective bioengineering methods. In this study, we analyzed the initial steps of mineral formation during secondary ossification of mouse femur based on biological and bioengineering approaches. We first found that some chondrocytes burst near the mineralized area. External factors that could trigger chondrocyte burst were then investigated. Chondrocyte burst was shown to be modulated by mechanical and osmotic pressure. A hypotonic solution, as well as mechanical stress, significantly induced chondrocyte burst. We further hypothesized that chondrocyte burst could be associated with space-making for mineral expansion. In fact, ex vivo culture of femur epiphysis in hypotonic conditions, or under mechanical pressure, enhanced mineral formation, compared to normal culture conditions. Additionally, the effect of mechanical pressure on bone formation in vivo was investigated by immobilization of mouse lower limbs to decrease the body pressure onto the joints. The results showed that limb immobilization suppressed bone formation. Together, these results suggest chondrocyte burst as a novel fate of chondrocytes, and that manipulation of chondrocyte burst with external mechano-chemical stimuli could be an additional approach for cartilage and bone tissue engineering.

Diagnosis and treatment of osteochondritis dissecans of the humeral capitellum.

Osteochondritis dissecans (OCD) of the humeral capitellum is a critical elbow injury in adolescent overhead throwing athletes. However, its etiology remains unknown. Medical examinations using ultrasonography found that the prevalence of capitellar OCD among adolescent baseball players was approximately from 1% to 3%. A plain anteroposterior radiograph with the elbow in 45° of flexion is essential for the diagnosis of an OCD lesion. The stability of OCD lesions is evaluated on plain radiographs, computed tomography, and magnetic resonance imaging (MRI). Imaging features of the unstable lesions are an epiphyseal closure of the capitellum or a lateral epicondyle, a displaced fragment, or irregular contours of the articular surface and a high signal interface on T2-weighted MRI. A stable lesion has the potential to be healed with conservative treatment. By contrast, surgical treatment should be considered if there is no radiographic improvement within 3 months. In addition, surgery should be performed for the lesions that cause pain during daily activities, have a locking phenomenon, or which are assessed by imaging as obviously unstable. Arthroscopic debridement/loose body removal can be performed for small lesions (≤12 mm in diameter). For large lesions (>12 mm), preservation and/or reconstruction of the articular surface should be selected, such as bone-peg fixation of the lateral part of the fragment and osteochondral autograft transplantation (OAT) from the knee. In the future directions, there is no comparative study of OAT from the knee and rib. In addition, little is known about its long-term outcome, or resulting osteoarthritis. A recent meta-analysis showed that grafts harvested from the knee may lead to donor site morbidity (7.8%). Thus, a novel cartilage tissue engineering approach is anticipated.

Analyzing Biological Performance of 3D-Printed, Cell-Impregnated Hybrid Constructs for Cartilage Tissue Engineering.

Three-dimensional (3D) bioprinting of hybrid constructs is a promising biofabrication method for cartilage tissue engineering because a synthetic polymer framework and cell-impregnated hydrogel provide structural and biological features of cartilage, respectively. During bioprinting, impregnated cells may be subjected to high temperatures (caused by the adjacent melted polymer) and process-induced mechanical forces, potentially compromising cell function. This study addresses these biofabrication issues, evaluating the heat distribution of printed polycaprolactone (PCL) strands and the rheological property and structural stability of alginate hydrogels at various temperatures and concentrations. The biocompatibility of parameters from these studies was tested by culturing 3D hybrid constructs bioprinted with primary cells from embryonic chick cartilage. During initial two-dimensional culture expansion of these primary cells, two morphologically and molecularly distinct cell populations ("rounded" and "fibroblastic") were isolated. The biological performance of each population was evaluated in 3D hybrid constructs separately. The cell viability, proliferation, and cartilage differentiation were observed at high levels in hybrid constructs of both cell populations, confirming the validity of these 3D bioprinting parameters for effective cartilage tissue engineering. Statistically significant performance variations were observed, however, between the rounded and fibroblastic cell populations. Molecular and morphological data support the notion that such performance differences may be attributed to the relative differentiation state of rounded versus fibroblastic cells (i.e., differentiated chondrocytes vs. chondroprogenitors, respectively), which is a relevant issue for cell-based tissue engineering strategies. Taken together, our study demonstrates that bioprinting 3D hybrid constructs of PCL and cell-impregnated alginate hydrogel is a promising approach for cartilage tissue engineering.

Platelet-rich plasma gel composited with nondegradable porous polyurethane scaffolds as a potential auricular cartilage alternative.

Total auricular reconstruction is still a challenge, and autologous cartilage transplant is the main therapy so far. Tissue engineering provides a promising method for auricular cartilage reconstruction. However, although degradable framework demonstrated excellent initial cosmetic details, it is difficult to maintain the auricular contour over time and the metabolites tended to be harmful to human body. In this study, biocompatible and safe nondegradable elastic polyurethane was used to make porous scaffold in specific details by rapid prototyping technology. Platelet-rich plasma contains fibrin and abundant autologous growth factors, which was used as cell carriers for invitro expanded cells. When crosslinking polyurethane framework, platelet-rich plasma and cells together, we successfully made polyurethane/platelet-rich plasma/cell composites, and implanted them into dorsal subcutaneous space of nude mice. The results showed that this method resulted in more even cell distribution and higher cell density, promoted chondrocyte proliferation, induced higher level expressions of aggrecan and type II collagen gene, increased content of newly developed glycosaminoglycans, and produced high-quality cartilaginous tissue. This kind of cartilage tissue engineering approach may be a potential promising alternative for external ear reconstruction.

Effect of chondrocyte-derived early extracellular matrix on chondrogenesis of placenta-derived mesenchymal stem cells

The extracellular matrix (ECM) surrounding cells contains a variety of proteins that provide structural support and regulate cellular functions. Previous studies have shown that decellularized ECM isolated from tissues or cultured cells can be used to improve cell differentiation in tissue engineering applications. In this study we evaluated the effect of decellularized chondrocyte-derived ECM (CDECM) on the chondrogenesis of human placenta-derived mesenchymal stem cells (hPDMSCs) in a pellet culture system. After incubation with or without chondrocyte-derived ECM in chondrogenic medium for 1 or 3 weeks, the sizes and wet masses of the cell pellets were compared with untreated controls (hPDMSCs incubated in chondrogenic medium without chondrocyte-derived ECM). In addition, histologic analysis of the cell pellets (Safranin O and collagen type II staining) and quantitative reverse transcription-PCR analysis of chondrogenic markers (aggrecan, collagen type II, and SOX9) were carried out. Our results showed that the sizes and masses of hPDMSC pellets incubated with chondrocyte-derived ECM were significantly higher than those of untreated controls. Differentiation of hPDMSCs (both with and without chondrocyte-derived ECM) was confirmed by Safranin O and collagen type II staining. Chondrogenic marker expression and glycosaminoglycan (GAG) levels were significantly higher in hPDMSC pellets incubated with chondrocyte-derived ECM compared with untreated controls, especially in cells precultured with chondrocyte-derived ECM for 7 d. Taken together, these results demonstrate that chondrocyte-derived ECM enhances the chondrogenesis of hPDMSCs, and this effect is further increased by preculture with chondrocyte-derived ECM. This preculture method for hPDMSC chondrogenesis represents a promising approach for cartilage tissue engineering.