Native Osteochondral Tissue Research Articles

Chitosan-Based Hierarchical Scaffolds Crosslinked with Genipin

Osteochondral defects present significant challenges for effective tissue regeneration due to the complex composition of bone and cartilage. To address this challenge, this study presents the fabrication of hierarchical scaffolds combining chitosan/β-tricalcium phosphate (β-TCP) to simulate a bone-like layer, interconnected with a silk fibroin layer to mimic cartilage, thus replicating the cartilage-like layer to mimic the native osteochondral tissue architecture. The scaffolds were produced by freeze-drying and then crosslinking with genipin. They have a crosslinking degree of up to 24%, which promotes a structural rearrangement and improved connection between the different layers. Micro-CT analysis demonstrated that the structures have distinct porosity values on their top layer (up to 84%), interface (up to 65%), and bottom layer (up to 77%) and are dependent on the concentration of β-tricalcium phosphate used. Both layers were confirmed to be clearly defined by the distribution of the components throughout the constructs, showing adequate mechanical properties for biomedical use. The scaffolds exhibited lower weight loss (up to 7%, 15 days) after enzymatic degradation due to the combined effects of genipin crosslinking and β-TCP incorporation. In vitro studies showed that the constructs supported ATDC5 chondrocyte-like cells and MC3T3 osteoblast-like cells in duo culture conditions, providing a suitable environment for cell adhesion and proliferation for up to 14 days. Overall, the physicochemical properties and biological results of the developed chitosan/β-tricalcium phosphate/silk fibroin bilayered scaffolds suggest that they may be potential candidates for osteochondral tissue strategies.

Bioreactor strategies for tissue-engineered osteochondral constructs: Advantages, present situations and future trends

The aim of osteochondral tissue engineering is to achieve the complex, functional and three-dimensional tissue regeneration under well defined, controlled and reproducible conditions in vitro. To achieve tissue-engineered products in vitro that incorporate rapidly in vivo with healthy tissue, it is essential to develop high-performance cell/scaffold culture systems that mimic the dynamics of the in vivo environment. Bioreactors could provide specific physicochemical culture environment, suitable mechanical stimulation and controlled condition for the development of osteochondral constructs in vitro. This review highlighted the multifunction of bioreactor in tissue engineering, and presented microenvironment and biomechanics of native osteochondral tissue, to illustrate the necessity of establishing osteochondral constructs by bioreactor. Then, we especially emphasized the advantages and limitations of various bioreactors. Furthermore, we systematically summarized and discussed the development of bioreactor-based production systems for bone, cartilage and osteochondral tissue engineering in recent years. Finally, we made a simple conclusion and offered perspectives of bioreactor‐based osteochondral tissue engineering. This review aims to serve as a reference for incorporating bioreactor strategies which could provide mechanical stimulation and physicochemical culture environment into the osteochondral construct culture regimens.

Characterization of Migratory Cells From Bioengineered Bovine Cartilage in a 3D Co-culture Model

Background: Chondrocyte migration in native cartilage is limited and has been implicated as one of the reasons for the poor integration of native implants. Through use of an in vitro integration model, it has previously been shown that cells from bioengineered cartilage can migrate into the native host cartilage during integration. Platelet-rich plasma (PRP) treatment further enhanced integration of bioengineered cartilage to native cartilage in vitro. However, it is not known how PRP treatment of the bioengineered construct promotes this. Hypothesis: PRP supports cell migration from bioengineered cartilage and these migratory cells have the ability to accumulate cartilage-like matrix. Study Design: Controlled laboratory study. Methods: Osteochondral-like constructs were generated by culturing primary bovine chondrocytes on the top surface of a porous bone substitute biomaterial composed of calcium polyphosphate. After 1 week in culture, the constructs were submerged in PRP and placed adjacent, but 2 mm distant, to a native bovine osteochondral plug in a co-culture model for 2 weeks. Cell migration was monitored using phase-contrast imaging. Cell phenotype was determined by evaluating the gene expression of matrix metalloprotease 13 (MMP-13), Ki67, and cartilage matrix molecules using quantitative polymerase chain reaction. When tissue formed, it was assessed by histology, immunohistochemistry, and quantification of matrix content. Results: PRP treatment resulted in the formation of a fiber network connecting the bioengineered cartilage and native osteochondral plug. Cells from both the bioengineered cartilage and the native osteochondral tissue migrated onto the PRP fibers and formed a tissue bridge after 2 weeks of culture. Migratory cells on the tissue bridge expressed higher levels of collagen types II and I (COL2, COL1), Ki67 and MMP-13 mRNA compared with nonmigratory cells in the bioengineered cartilage. Ki67 and MMP-13–positive cells were found on the edges of the tissue bridge. The tissue bridge accumulated COL1 and COL2 and aggrecan and contained comparable collagen and glycosaminoglycan content to the bioengineered cartilage matrix. The tissue bridge did not reliably develop in the absence of cells from the native osteochondral plug. Conclusion: Bioengineered cartilage formed by bovine chondrocytes contains cells that can migrate on PRP fibers and form cartilaginous tissue. Clinical Relevance: Migratory cells from bioengineered cartilage may promote cartilage integration. Further studies are required to determine the role of migratory cells in integration in vivo.

3D Bioprinting of Biomimetic Bilayered Scaffold Consisting of Decellularized Extracellular Matrix and Silk Fibroin for Osteochondral Repair.

Recently, three-dimensional (3D) bioprinting technology is becoming an appealing approach for osteochondral repair. However, it is challenging to develop a bilayered scaffold with anisotropic structural properties to mimic a native osteochondral tissue. Herein, we developed a bioink consisting of decellularized extracellular matrix and silk fibroin to print the bilayered scaffold. The bilayered scaffold mimics the natural osteochondral tissue by controlling the composition, mechanical properties, and growth factor release in each layer of the scaffold. The in vitro results show that each layer of scaffolds had a suitable mechanical strength and degradation rate. Furthermore, the scaffolds encapsulating transforming growth factor-beta (TGF-β) and bone morphogenetic protein-2 (BMP-2) can act as a controlled release system and promote directed differentiation of bone marrow-derived mesenchymal stem cells. Furthermore, the in vivo experiments suggested that the scaffolds loaded with growth factors promoted osteochondral regeneration in the rabbit knee joint model. Consequently, the biomimetic bilayered scaffold loaded with TGF-β and BMP-2 would be a promising strategy for osteochondral repair.

Current researches on design and manufacture of biopolymer-based osteochondral biomimetic scaffolds

Currently, osteochondral (OC) tissue engineering has become a potential treatment strategy in repairing chondral lesions and early osteoarthritis due to the limited self-healing ability of cartilage. However, it is still challenging to ensure the integrity, physiological function and regeneration ability of stratified OC scaffolds in clinical application. Biomimetic OC scaffolds are attractive to overcome the above problems because of their similar biological and mechanical properties with native OC tissue. As a consequence, the researches on biomimetic design to achieve the tissue function of each layer, and additive manufacture (AM) to accomplish composition switch and ultrastructure of personalized OC scaffolds have made a remarkable progress. In this review, the design methods of biomaterial and structure as well as computer-aided design, and performance prediction of biopolymer-based OC scaffolds are presented; then, the characteristics and limitations of AM technologies and the integrated manufacture schemes in OC tissue engineering are summarized; finally, the novel biomaterials and techniques and the inevitable trends of multifunctional bio-manufacturing system are discussed for further optimizing production of tissue engineering OC scaffolds.

Bioinspired stratified electrowritten fiber-reinforced hydrogel constructs with layer-specific induction capacity for functional osteochondral regeneration

Despite significant advances in osteochondral tissue engineering, it remains challenging to successfully reconstruct native-like complex tissues organized in three-dimension with spatially varying compositional, structural and functional properties. In this contribution, inspired by the gradients in extracellular matrix (ECM) composition and collagen fiber architecture in native osteochondral tissue, we designed and fabricated a tri-layered (superficial cartilage (S), deep cartilage (D) and subchondral bone (B) layer) stratified scaffold in which a mesenchymal stem cell (MSC)-laden gelatin methacrylamide (GelMA) hydrogel with zone-specific growth factor delivery was combined with melt electrowritten triblock polymer of poly(ε-caprolactone) and poly(ethylene glycol) (PCEC) networks with depth-dependent fiber organization. Introducing PCEC fibers into the weak GelMA hydrogel contributed to a significant increase in mechanical strength. In vitro biological experiments indicated that the stratified fiber-reinforced and growth factor-loaded hydrogel construct induced the MSCs to differentiate down both the chondrogenic and osteogenic lineages and that the engineered complex exhibited cellular phenotype and matrix accumulation profiles resembling those of the native tissue. Simultaneous cartilage and subchondral bone regeneration were achieved in vivo by using the tri-layered integrated scaffold. More importantly, the inclusion of the S layer could impart the regenerated cartilage with a more lubricating and wear-resistant surface. These findings suggest that the bioinspired construct mimicking the spatial variations of native osteochondral tissue might serve as a promising candidate to enhance osteochondral regeneration.

The development of natural polymer scaffold-based therapeutics for osteochondral repair.

Due to the limited regenerative capacity of cartilage, untreated joint defects can advance to more extensive degenerative conditions such as osteoarthritis. While some biomaterial-based tissue-engineered scaffolds have shown promise in treating such defects, no scaffold has been widely accepted by clinicians to date. Multi-layered natural polymer scaffolds that mimic native osteochondral tissue and facilitate the regeneration of both articular cartilage (AC) and subchondral bone (SCB) in spatially distinct regions have recently entered clinical use, while the transient localized delivery of growth factors and even therapeutic genes has also been proposed to better regulate and promote new tissue formation. Furthermore, new manufacturing methods such as 3D bioprinting have made it possible to precisely tailor scaffold micro-architectures and/or to control the spatial deposition of cells in requisite layers of an implant. In this way, natural and synthetic polymers can be combined to yield bioactive, yet mechanically robust, cell-laden scaffolds suitable for the osteochondral environment. This mini-review discusses recent advances in scaffolds for osteochondral repair, with particular focus on the role of natural polymers in providing regenerative templates for treatment of both AC and SCB in articular joint defects.

Chitosan/collagen based biomimetic osteochondral tissue constructs: A growth factor-free approach

Tissue engineering approach offers alternative strategies to develop multi-layered/multi-component osteochondral mimetic constructs to meet the requirements of the heterogeneous and layered structure of native osteochondral tissue. Herein, an iterative overlaying process to fabricate a multi-layered scaffold with a gradient composition and layer specific structure have been developed by combining the natural extracellular matrix (ECM) components-chitosan, type I collagen, type II collagen, nanohydroxyapatite- of the osteochondral tissue in biomimetic compositions. Subchondral bone layer was prepared by using freeze-drying method to obtain 3D porous scaffolds. The calcified cartilage and cartilage layers were prepared by thermal gelation method in the hydrogel form. Osteochondral scaffolds fabricated by iterative overlaying of each distinct layer exhibited a porous, continuous gradient structure and supported cell proliferation in a co-culture of MC3T3-E1 preosteoblasts and ATDC5 chondrocytes. Histology and biochemical analysis showed enhanced extracellular matrix production and demonstrated collagen and glycosaminoglycan deposition. Expression of genes specific for bone, calcified cartilage and cartilage were improved in the osteochondral scaffold. Overall, these findings suggest that iterative overlaying of freeze-dried scaffolds and hydrogel matrices prepared by using ECM components in biomimetic ratios to fabricate gradient, multi-layered structures can be a promising strategy without the need for growth factors.

Role of the Calcified Cartilage Layer of an Integrated Trilayered Silk Fibroin Scaffold Used to Regenerate Osteochondral Defects in Rabbit Knees.

The repair of osteochondral defects remains challenging, given the complexity of native osteochondral tissue and the limited self-repair capacity of cartilage. Osteochondral tissue engineering is a promising strategy. Here, we fabricated a biomimetic osteochondral scaffold using silk fibroin and hydroxyapatite, including a calcified cartilage layer (CCL). We studied the role played by the CCL in terms of cell viability in vivo. We established osteochondral defects in rabbit knees to investigate the effects of CCL-containing scaffolds with or without adipose tissue-derived stem cells (ADSCs). We evaluated osteochondral tissue regeneration by calculating gross observational scores, via histological and immunohistochemical assessments, by performing quantitative biochemical and biomechanical analyses of new osteochondral tissue, and via microcomputed tomography of new bone at 4, 8, and 12 weeks after surgery. In terms of surface roughness and integrity, the CCL + ADSCs group was better than the CCL and the non-CCL + ADSCs groups at all time points tested; the glycosaminoglycan and collagen type II levels of the CCL + ADSCs group were highest, reflecting the important role played by the CCL in cartilage tissue repair. Subchondral bone smoothness was better in the CCL + ADSCs group than in the non-CCL + ADSCs and CCL groups. The CCL promoted smooth subchondral bone regeneration but did not obviously affect bone strength or quality. In conclusion, a biomimetic osteochondral scaffold with a CCL, combined with autologous ADSCs, satisfactorily regenerated a rabbit osteochondral defect. The CCL enhances cartilage and subchondral bone regeneration.

Hierarchical scaffolds for osteochondral tissue engineering

Osteochondral defects caused by various factors are still difficult clinical problems. With the development of tissue engineering, the strategies and methods for repairing osteochondral defects in the past decade have made great progress, and some osteochondral tissue stratified stent products have gradually entered the clinical trial stage.. The related articles on tissue engineering for the treatment of osteochondral defects were retrieved by searching databases with key words osteochondral defects, cartilage repair and hierarchical scaffolds. This paper discussed the research status of hierarchical scaffolds in osteochondral tissue engineering during recent five years. In this work, the classification of hierarchical scaffold including monophasic scaffolds, bilayered scaffolds, multilayered scaffolds and gradient scaffolds, are summarized by comparing different experiment researches. Furthermore, the advantages and disadvantages of different types of hierarchical scaffolds were introduced through analyzing relevant studies. Monophasic scaffolds can support the adhesion and proliferation of osteoblasts and chondrocytes, but lack the inherent stratified structure features required for osteochondral regeneration.. Bilayered scaffolds consist of a chondral layer and subchondral layer which base on the biocompatibility of monophasic scaffolds. Biphasic scaffolds are significantly better than monophasic scaffolds in simulating natural cartilage, but the interface between chondral and subchondral layer is poor binding. Compared with bilayered scaffold, trilayered scaffolds are added with an intermediate layer which simulates the calcification of normal cartilage between the two layers, so as to obtain better connection of the bone and cartilage layer. Unlike hierarchical scaffolds, gradient scaffolds provide a gradient connection between the layers, which is more similar to the native osteochondral tissue. In the past five years, the development of osteochondral layered scaffolds mainly depended on the novel structure and fabrication methods of scaffolds. However, correlational clinical studies are quite few. Further high quality and large clinical studies are still required.

Mechanical properties of hybrid triphasic scaffolds for osteochondral tissue engineering

Reproducing the advanced complexity of native tissue by means of the 3D multi-functional construct is a promising tissue engineering approach to osteochondral tissue regeneration. In this study, we present a porous 3D construct composed of three zones responsible for the regeneration of non-calcified cartilage, calcified cartilage and subchondral bone. These three zones of the hybrid were composed of modified biopolymers: (i) alginate (Alg) reinforced by short polylactide (PLA) fibres, (ii) alginate and gelatine methacrylate (GelMA) combined with ß-tricalcium phosphate particles (TCP), (iii) 3D printed polycaprolactone scaffold subsequently modified with the use of an innovative solvent treatment method based on acetone and ultrasound stimulation, respectively. Combining the advanced deposition systems based on: (i) 3D printing coupled with a spray crosslinking system, (ii) an innovative deposition system based on a coaxial-needle extruder, (iii) fused deposition modelling (FDM) connected with post-fabrication treatment, allows us to fabricate the triphasic construct that emulates the structure and properties of the native osteochondral tissue. The aim of the study was to investigate the mechanical properties of the fabricated hybrid and its individual zones. Our results demonstrate the load-bearing capabilities of TC, but nevertheless it should be implanted below the surface line of host cartilage to protect it from strong stresses, at the same time allowing native host tissues to grow into it.

Bilayered Scaffold Prepared from a Kartogenin-Loaded Hydrogel and BMP-2-Derived Peptide-Loaded Porous Nanofibrous Scaffold for Osteochondral Defect Repair.

Recently, a bilayered scaffold with an anisotropic structure mimicking a native osteochondral tissue shows considerable potential for treating osteochondral defects. Herein, a bilayered scaffold consisting of biomimetic cartilage and a subchondral bone architecture was constructed for repairing osteochondral defect. A hydrogel prepared by the Schiff base reaction of gelatin, silk fibroin, and oxidized dextran was designed as the cartilage layer, while a nanofibrous scaffold with a macroporous structure prepared from the polymer blend of poly(l-lactic acid)/poly(lactic-co-glycolic acid)/poly(ε-caprolactone) using the dual phase separation technique served as a subchondral layer. The subchondral layer was then treated with polydopamine coating for osteogenic factor immobilization. To facilitate the chondrogenic and osteogenic differentiation of bone marrow mesenchymal stem cells on the bilayered scaffold, the cartilage-inducing drug kartogenin (KGN) and osteogenic-inducing factor bone morphogenetic protein 2-derived peptides (P24 peptides) were, respectively, loaded on the subchondral layer. Next, the in vitro release of KGN and P24 peptide from the corresponding layer was monitored, respectively, and the results showed that both the release time of KGN and P24 peptides would last for more than 28 days. The in vitro results indicated that the KGN-loaded cartilage layer and P24 peptides-loaded subchondral layer were capable of supporting cell proliferation, and induced the chondrogenic and osteogenic differentiation, respectively. Furthermore, the in vivo experiments suggested that the bilayered scaffold significantly accelerated the regeneration of the osteochondral tissue in the rabbit knee joint model. Consequently, this bilayered scaffold loaded with KGN and P24 peptides would be a promising candidate for repairing osteochondral defect.

Fabrication and mechanical characterization of 3D printed vertical uniform and gradient scaffolds for bone and osteochondral tissue engineering

Recent developments in 3D printing (3DP) research have led to a variety of scaffold designs and techniques for osteochondral tissue engineering; however, the simultaneous incorporation of multiple types of gradients within the same construct remains a challenge. Herein, we describe the fabrication and mechanical characterization of porous poly(ε-caprolactone) (PCL) and PCL-hydroxyapatite (HA) scaffolds with incorporated vertical porosity and ceramic content gradients via a multimaterial extrusion 3DP system. Scaffolds of 0 wt% HA (PCL), 15 wt% HA (HA15), or 30 wt% HA (HA30) were fabricated with uniform composition and porosity (using 0.2 mm, 0.5 mm, or 0.9 mm on-center fiber spacing), uniform composition and gradient porosity, and gradient composition (PCL-HA15-HA30) and porosity. Micro-CT imaging and porosity analysis demonstrated the ability to incorporate both vertical porosity and pore size gradients and a ceramic gradient, which collectively recapitulate gradients found in native osteochondral tissues. Uniaxial compression testing demonstrated an inverse relationship between porosity, ϕ, and compressive modulus, E, and yield stress, σy, for uniform porosity scaffolds, however, no differences were observed as a result of ceramic incorporation. All scaffolds demonstrated compressive moduli within the appropriate range for trabecular bone, with average moduli between 86 ± 14–220 ± 26 MPa. Uniform porosity and pore size scaffolds for all ceramic levels had compressive moduli between 205 ± 37–220 ± 26 MPa, 112 ± 13–118 ± 23 MPa, and 86 ± 14–97 ± 8 MPa respectively for porosities ranging between 14 ± 4–20 ± 6%, 36 ± 3–43 ± 4%, and 54 ± 2–57 ± 2%, with the moduli and yield stresses of low porosity scaffolds being significantly greater (p < 0.05) than those of all other groups. Single (porosity) gradient and dual (composition/porosity) gradient scaffolds demonstrated compressive properties similar (p > 0.05) to those of the highest porosity uniform scaffolds (porosity gradient scaffolds 98 ± 23–107 ± 6 MPa, and 102 ± 7 MPa for dual composition/porosity gradient scaffolds), indicating that these properties are more heavily influenced by the weakest section of the gradient. The compression data for uniform scaffolds were also readily modeled, yielding scaling laws of the form E ∼ (1 − ϕ)1.27 and σy ∼ (1 − ϕ)1.37, which demonstrated that the compressive properties evaluated in this study were well-aligned with expectations from previous literature and were readily modeled with good fidelity independent of polymer scaffold geometry and ceramic content. All uniform scaffolds were similarly deformed and recovered despite different porosities, while the large-pore sections of porosity gradient scaffolds were significantly more deformed than all other groups, indicating that porosity may not be an independent factor in determining strain recovery. Moving forward, the technique described here will serve as the template for more complex multimaterial constructs with bioactive cues that better match native tissue physiology and promote tissue regeneration. Statement of significanceThis manuscript describes the fabrication and mechanical characterization of “dual” porosity/ceramic content gradient scaffolds produced via a multimaterial extrusion 3D printing system for osteochondral tissue engineering. Such scaffolds are designed to better address the simultaneous gradients in architecture and mineralization found in native osteochondral tissue. The results of this study demonstrate that this technique may serve as a template for future advances in 3D printing technology that may better address the inherent complexity in such heterogeneous tissues.

Open Reduction and Internal Fixation for the Treatment of Symptomatic Osteochondritis Dissecans of the Femoral Head in Patients With Sequelae of Legg-Calvé-Perthes Disease.

Osteochondritis dissecans (OCD) is estimated to occur in 2% to 7% of patients with Legg-Calvé-Perthes disease (LCPD). Unstable osteochondral fragments secondary to LCPD may produce mechanical symptoms requiring surgical intervention. Reattachment of the fragment with open reduction and internal fixation (ORIF) may provide good clinical outcomes. The purpose of this study is to report short-term clinical and radiographic results of ORIF for the treatment of symptomatic osteochondral lesions resulting from LCPD. Clinical data including patient demographics and patient-reported outcome scores were collected prospectively. All patients underwent preoperative radiographs and magnetic resonance imaging confirming an unstable OCD fragment as well as postoperative radiographs at regular intervals. Indications for ORIF of the OCD fragment were hip pain and mechanical symptoms with radiographic evidence of LCPD and a magnetic resonance imaging demonstrating an OCD fragment of the femoral head. Osteochondral fragment instability was confirmed intraoperatively. Preoperative and postoperative physical examination findings were documented. All patients failed a course of nonsurgical treatment (activity modification, anti-inflammatories, and physical therapy). From a total of 64 consecutive patients treated with hip preservation surgery for LCPD, 7 patients with symptomatic OCD secondary to LCPD were treated with surgical hip dislocation and ORIF of the femoral head osteochondral fragment. OCD size lesion ranged from 200 to 625 mm. All patients reported marked clinical improvement, with resolution of both pain and mechanical symptoms. Radiographs at final follow-up demonstrated complete osteochondral fragment healing without implant failure. Mean follow-up was 4.6 years (range, 1.1 to 7.4 y). There was a significant postoperative improvement in measured internal rotation in flexion (5.0±5.0 to 16.4±9.8; P=0.02). Modified Harris Hip Score markedly improved from baseline to final follow-up (47.8 to 82.7; [INCREMENT]34.9; minimal clinically important difference, 11; P=0.002), with all patients meeting minimal clinically important difference for modified Harris Hip Score. There were no complications and no progression of osteoarthritis in all patients at final follow-up. We have demonstrated both predictable radiographic healing and marked clinical improvement following ORIF of symptomatic post-Perthes OCD lesions. We advocate ORIF for symptomatic osteochondral lesions as a first-line surgical treatment for these patients due to the advantages of native osteochondral tissue preservation, predictable healing, and marked clinical improvement. Level IV-case series.

Nanomaterials/Nanocomposites for Osteochondral Tissue.

For many years, the avascular nature of cartilage tissue has posed a clinical challenge for replacement, repair, and reconstruction of damaged cartilage within the human body. Injuries to cartilage and osteochondral tissues can be due to osteoarthritis, sports, aggressive cancers, and repetitive stresses and inflammation on wearing tissue. Due to its limited capacity for regeneration or repair, there is a need for suitable material systems which can recapitulate the function of the native osteochondral tissue physically, mechanically, histologically, and biologically. Tissue engineering (TE) approaches take advantage of principles of biomedical engineering, clinical medicine, and cell biology to formulate, functionalize, and apply biomaterial scaffolds to aid in the regeneration and repair of tissues. Nanomaterial science has introduced new methods for improving and fortifying TE scaffolds, and lies on the forefront of cutting-edge TE strategies. These nanomaterials enable unique properties directly correlated to their sub-micron dimensionality including structural and cellular advantages. Examples include electrospun nanofibers and emulsion nanoparticles which provide nanoscale features for biomaterials, more closely replicating the 3D extracellular matrix, providing better cell adhesion, integration, interaction, and signaling. This chapter aims to provide a detailed overview of osteochondral regeneration and repair using TE strategies with a focus on nanomaterials and nanocomposites.

Thermogel-Coated Poly(ε-Caprolactone) Composite Scaffold for Enhanced Cartilage Tissue Engineering

A three-dimensional (3D) composite scaffold was prepared for enhanced cartilage tissue engineering, which was composed of a poly(ε-caprolactone) (PCL) backbone network and a poly(lactide-co-glycolide)-block-poly(ethylene glycol)-block-poly(lactide-co-glycolide) (PLGA–PEG–PLGA) thermogel surface. The composite scaffold not only possessed adequate mechanical strength similar to native osteochondral tissue as a benefit of the PCL backbone, but also maintained cell-friendly microenvironment of the hydrogel. The PCL network with homogeneously-controlled pore size and total pore interconnectivity was fabricated by fused deposition modeling (FDM), and was impregnated into the PLGA–PEG–PLGA solution at low temperature (e.g., 4 °C). The PCL/Gel composite scaffold was obtained after gelation induced by incubation at body temperature (i.e., 37 °C). The composite scaffold showed a greater number of cell retention and proliferation in comparison to the PCL platform. In addition, the composite scaffold promoted the encapsulated mesenchymal stromal cells (MSCs) to differentiate chondrogenically with a greater amount of cartilage-specific matrix production compared to the PCL scaffold or thermogel. Therefore, the 3D PCL/Gel composite scaffold may exhibit great potential for in vivo cartilage regeneration.

Biomimetic multidirectional scaffolds for osteochondral repair by sequential freeze casting

Event Abstract Back to Event Biomimetic multidirectional scaffolds for osteochondral repair by sequential freeze casting Drew Clearfield1 and Mei Wei1 1 University of Connecticut, Department of Materials Science and Engineering, Institute of Materials Science, United States Introduction: By the year 2030, osteoarthritis is expected to affect 67 million Americans[1]. Osteoarthritis commonly leads to the creation of osteochondral lesions[2]. These lesions disrupt the unique zonal organization of osteochondral extracellular matrix, which proves vital to its function[3]. There exists a need for tissue engineering strategies to induce the regeneration of new tissue that recapitulates this zonal organization and corresponding functionality. In the current work, a sequential freeze casting process is detailed that allows for the fabrication of multi-zonal collagen-based scaffolds that mimic three zonal features of native osteochondral extracellular matrix: the superficial zone (SZ), the transition zone (TZ), and the deep zone (DZ) morphologies [1], zonal pore size gradient [2], and zone-specific compositions [3] of articular cartilage and subchondral bone in a single construct. To mimic the zonal structure, a multizonal scaffold has been designed through a novel sequential freezing process for osteochondral defect repair. Zonal anisotropy is created through two stages of unidirectional freezing. Zone-specific pore size is controlled through applied cooling rate and zone-specific composition is controlled by adjusting initial suspension formulations. Methods: Sequential freeze casting was performed to create multizonal collagen (Col) and collagen-hyaluronic acid (Col-HYA)/collagen-hydroxyapatite (Col-HA) composite scaffolds. For collagen-only scaffolds, 2 wt% type I collagen was homogenized in deionized water, degased, and unidirectionally frozen. The frozen structure was flipped 90º, partially melted, and a second collagen suspension was loaded and unidirectionally frozen atop the first. Sequentially frozen scaffolds were lyophilized at -40ºC for 3 days, crosslinked with 2% EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride):NHS (N-hydroxysuccinimide) (1:0.25 molar ratio), and lyophilized for another 3 days. The effect of cooling rate on zonal pore size was investigated. SEM analysis was performed to view zonal morphologies (longitudinal sections) and quantify pore sizes (transverse sections). To further mimic the zonal composition of native osteochondral extracellular matrix, HYA and HA was added to the collagen suspensions and the scaffolds were prepared using the approached described above. A Col-HYA (1.8 wt% Col, 0.2 wt% HYA) suspension was first unidirectionally frozen at 2ºC/min. A second Col-HYA slurry of increased HYA and decreased Col content was frozen 90º to the first at 0.5ºC/min, where a third Col-HA slurry was layered atop. Results and Discussion: SEM analysis reveals three distinct zones of collagen fiber orientation seamlessly integrated together (Figure 1). A 200-300 µm transition region of isotropic collagen integrates between two zones of unidirectionally aligned fibers, perpendicular to one another. Pore size quantification revealed a trend of increasing pore sizes through the depth of the scaffold. Zonal pore sizes were controlled by the applied cooling rate. Quicker cooling led to the formation of smaller pores, while slower cooling promoted larger pores. Fig. 1: Scanning electron micrograph of collagen-based multizonal scaffolds. SZ - superficial zone, TZ - transition zone, DZ - deep zone. Conclusions: Sequentially freeze cast multizonal collagen-based scaffolds were fabricated that mimic the zone-specific collagen fiber orientation, pore size, and composition present in native osteochondral tissue. The authors would like to thank NSF grants (CBET-1133883 and CBET-1347130) for their support

Integrated trilayered silk fibroin scaffold for osteochondral differentiation of adipose-derived stem cells.

Repairing osteochondral defects (OCD) remains a formidable challenge due to the high complexity of native osteochondral tissue and the limited self-repair capability of cartilage. Osteochondral tissue engineering is a promising strategy for the treatment of OCD. In this study, we fabricated a novel integrated trilayered scaffold using silk fibroin and hydroxyapatite by combining paraffin-sphere leaching with a modified temperature gradient-guided thermal-induced phase separation (TIPS) technique. This biomimetic scaffold is characterized by three layers: a chondral layer with a longitudinally oriented microtubular structure, a bony layer with a 3D porous structure and an intermediate layer with a dense structure. Live/dead and CCK-8 tests indicated that this scaffold possesses good biocompatibility for supporting the growth, proliferation, and infiltration of adipose-derived stem cells (ADSCs). Histological and immunohistochemical stainings and real-time polymerase chain reaction (RT-PCR) confirmed that the ADSCs could be induced to differentiate toward chondrocytes or osteoblasts in vitro at chondral and bony layers in the presence of chondrogenic- or osteogenic-induced culture medium, respectively. Moreover, the intermediate layer could play an isolating role for preventing the cells within the chondral and bony layers from mixing with each other. In conclusion, the trilayered and integrated osteochondral scaffolds can effectively support cartilage and bone tissue generation in vitro and are potentially applicable for OC tissue engineering in vivo.

Osteochondral tissue regeneration through polymeric delivery of DNA encoding for the SOX trio and RUNX2

Native osteochondral repair is often inadequate owing to the inherent properties of the tissue, and current clinical repair strategies can result in healing with a limited lifespan and donor site morbidity. This work investigates the use of polymeric gene therapy to address this problem by delivering DNA encoding for transcription factors complexed with the branched poly(ethylenimine)–hyaluronic acid (bPEI–HA) delivery vector via a porous oligo[poly(ethylene glycol) fumarate] hydrogel scaffold. To evaluate the potential of this approach, a bilayered scaffold mimicking native osteochondral tissue organization was loaded with DNA/bPEI–HA complexes. Next, bilayered implants either unloaded or loaded in a spatial fashion with bPEI–HA and DNA encoding for either Runt-related transcription factor 2 (RUNX2) or SRY (sex determining region Y)-box 5, 6, and 9 (the SOX trio), to generate bone and cartilage tissues respectively, were fabricated and implanted in a rat osteochondral defect. At 6weeks post-implantation, micro-computed tomography analysis and histological scoring were performed on the explants to evaluate the quality and quantity of tissue repair in each group. The incorporation of DNA encoding for RUNX2 in the bone layer of these scaffolds significantly increased bone growth. Additionally, a spatially loaded combination of RUNX2 and SOX trio DNA loading significantly improved healing relative to empty hydrogels or either factor alone. Finally, the results of this study suggest that subchondral bone formation is necessary for correct cartilage healing.

Technologies for Multilayered Scaffolds Suitable for Interface Tissue Engineering

Multilayered scaffolds that provide tailored space‐specific biological and mechanical functions are promising for interface tissue engineering such as in osteochondral tissue regeneration. In this study, fabrication techniques, including foam replication, gelation, and freeze‐drying techniques, were combined in order to manufacture stratified scaffolds mimicking the layered structure of native osteochondral tissue. 45S5 Bioglass® and alginate were used to fabricate 3D highly porous (composite) scaffolds for the underlying subchondral bone layer. Freeze‐dried alginate‐based scaffolds were produced for the cartilage layer. Finally, both layers were integrated using a novel alginate/45S5 Bioglass® hybrid interface acting as an adhesive, which functions as the cartilage–bone interfacial layer. Novel multilayered scaffolds were optimized to achieve the complex requirements for osteochondral tissue engineering such as the 3D architecture, porous structure, physical, and mechanical properties, which are presented and discussed in the context of the intended application of the novel scaffolds in osteochondral tissue regeneration.