Neocartilage Formation Research Articles

TP8, A Novel Chondroinductive Peptide, Significantly Promoted Neo-Cartilage Repair without Activating Bone Formation.

The repair of large cartilage defects remains highly challenging in the fields of orthopedics and oral and maxillofacial surgery. A chondroinductive agent is promising to activate endogenous mesenchymal stem cells (MSCs) so as to facilitate cartilage regeneration. In this study, we analyzethe crystallographic data of the critical binding domain of transforming growth factor β3 (TGF-β3) with its type II receptor and successfully develop a novel chondroinductive peptide - TGF-β3-derived peptide No. 8 (TP8) that can induce an ectopic cartilage formation without obvious bone formation. TP8 shows a comparable capacity as TGF-β3 in enhancing glycosaminoglycans (GAGs) and proteoglycans (PGs) secretion in the micromass of bone marrow MSCs (BMSCs) and promoting the expression of chondrogenic markers in comparison with the Control group. TP8 induces a significantly higher expression of the SRY-box transcription factor 9 (Sox9) gene than TGF-β3. Moreover, TP8 significantly upregulates the phosphorylation of Smad1/5 but not MAPK/JNK or Smad 2/3. The knockdown of low-density lipoprotein receptor (LDLR) -related protein-1 (Lrp1), a transmembrane endocytosis receptor, nullifies the TP8-induced Sox9 expression. In the critical-size cartilage defects in rabbit medial femoral condyles, TP8 can induce neo-cartilage formation with a significantly thicker deep zone in comparison with the TGF-β3 and Control. These findings suggest a promising application potential of TP8 in cartilage tissue engineering.

AMIC achieves sustained clinical improvement in isolated patellar cartilage defects over 5 years, correlating with MRI.

To evaluate 5-year postoperative clinical outcomes of autologous matrix-induced chondrogenesis (AMIC) for isolated ICRS grade 3-4 patellar cartilage defects and correlate outcomes with magnetic resonance imaging (MRI). The hypothesis was that AMIC would improve clinical symptoms and induce neocartilage formation, visible on MRI, making it a safe and effective option for repairing focal patellar cartilage defects. The cohort comprised 13 focal patellar lesions in 12 patients. Pain visual analogue scale (VAS), Knee Injury and Osteoarthritis Outcome Score (KOOS), Kujala score, EuroQol-5D Health Survey questionnaire and MRI data were assessed preoperatively and at 2 and 5 years postoperatively. All MRI scans were evaluated using the Magnetic Resonance Observation of Cartilage Repair Tissue System. Descriptive statistics were calculated on all data. Inferential analysis comparing outcome scores before and after surgery employed the nonparametric Wilcoxon signed-rank test, with the nonparametric Friedman test used to detect differences across multiple test attempts. p < 0.05 was considered statistically significant. Twelve patients (23-52 years old) with patellofemoral chondral full-thickness defects (2-4 cm2) were treated. At a 5-year follow-up, eleven knees showed MRI improvement. Two were asymptomatic and nine showed clear clinical improvement. Only one knee showed no clinical improvement. MRI revealed a defect filling with newly formed cartilage characterized by a less compact and heterogeneous signal. Cartilage degradation or joint damage was observed in two knees, and bone formation within the plate was identified in four. AMIC significantly improved patients' VAS pain, KOOS, EuroQol-5D and Kujala scores compared to preoperative baseline for up to 5 years postoperatively. Satisfactory clinical outcomes and new cartilage formation, as observed by MRI, are achieved with AMIC at mid-term follow-up for ICRS grade 3-4 in small-to-medium-sized patellar defects in patients under 52 years of age, with improvements maintained for up to 5 years. Level III.

Cartilage tissue engineering using decellularized biomatrix hydrogel containing TGF-β-loaded alginate microspheres in mechanically loaded bioreactor

Physiochemical tissue inducers and mechanical stimulation are both efficient variables in cartilage tissue fabrication and regeneration. In the presence of biomolecules, decellularized extracellular matrix (ECM) may trigger and enhance stem cell proliferation and differentiation. Here, we investigated the controlled release of transforming growth factor beta (TGF-β1) as an active mediator of mesenchymal stromal cells (MSCs) in a biocompatible scaffold and mechanical stimulation for cartilage tissue engineering. ECM-derived hydrogel with TGF-β1-loaded alginate-based microspheres (MSs) was created to promote human MSC chondrogenic development. Ex vivo explants and a complicated multiaxial loading bioreactor replicated the physiological conditions. Hydrogels with/without MSs and TGF-β1 were highly cytocompatible. MSCs in ECM-derived hydrogel containing TGF-β1/MSs showed comparable chondrogenic gene expression levels as those hydrogels with TGF-β1 added in culture media or those without TGF-β1. However, constructs with TGF-β1 directly added within the hydrogel had inferior properties under unloaded conditions. The ECM-derived hydrogel group including TGF-β1/MSs under loading circumstances formed better cartilage matrix in an ex vivo osteochondral defect than control settings. This study demonstrates that controlled local delivery of TGF-β1 using MSs and mechanical loading is essential for neocartilage formation by MSCs and that further optimization is needed to prevent MSC differentiation towards hypertrophy.

In vitro and in vivo investigation of chitosan/silk fibroin injectable interpenetrating network hydrogel with microspheres for cartilage regeneration

Articular cartilage is an avascular and almost acellular tissue with limited self-regenerating capabilities. Although injectable hydrogels have garnered a lot of attention as a promising treatment, a biocompatible hydrogel with adequate mechanical properties is yet to be created. In this study, an interpenetrating network hydrogel comprised of chitosan and silk fibroin was created through electrostatic and hydrophobic bonds, respectively. The polymeric network of the scaffold combined an effective microenvironment for cell activity with enhanced mechanical properties to address the current issues in cartilage scaffolds. Furthermore, microspheres (MS) were utilized for a controlled release of methylprednisolone acetate (MPA), around ~75 % after 35 days. The proposed scaffolds demonstrated great mechanical stability with ~0.047 MPa compressive moduli and ~145 kPa compressive strength. Moreover, the degradation rate of the samples (~45 % after 35 days) was optimized to match neo-cartilage formation. Furthermore, the use of natural biomaterials yielded good biocompatibility with ~76 % chondrocyte viability after 7 days. According to gross observation after 12 weeks the defect site of the treated groups was filled with minimally discernible boundary. These results were confirmed by histopathology assays were the treated groups showed higher chondrocyte count and collagen type II expression.

Mind the Viscous Modulus: The Mechanotransductive Response to the Viscous Nature of Isoelastic Matrices Regulates Stem Cell Chondrogenesis.

The design of hydrogels as mimetics of tissues' matrices typically disregards the viscous nature of native tissues and focuses only on their elastic properties. In the case of stem cell chondrogenesis, this has led to contradictory results, likely due to unreported changes in the matrices' viscous modulus. Here, by employing isoelastic matrices with Young's modulus of ≈12kPa, variations in viscous properties alone (i.e., loss tangent between 0.1 and 0.25) are demonstrated to be sufficient to drive efficient growth factor-free chondrogenesis of human mesenchymal stem cells, both in 2D and 3D cultures. The increase of the viscous component of RGD-functionalized polyacrylamide or polyethylene glycol maleimide hydrogels promotes a phenotype with reduced adhesion, alters mechanosensitive signaling, and boosts cell-cell contacts. In turn, this upregulates the chondrogenic transcription factor SOX9 and supports neocartilage formation, demonstrating that the mechanotransductive response to the viscous nature of the matrix can be harnessed to direct cell fate.

Full-thickness osteochondral defect repair using a biodegradable bilayered scaffold of porous zinc and chondroitin sulfate hydrogel

The regeneration of osteochondral tissue necessitates the re-establishment of a gradient owing to the unique characteristics and healing potential of the chondral and osseous phases. As the self-healing capacity of hyaline cartilage is limited, timely mechanical support during neo-cartilage formation is crucial to achieving optimal repair efficacy. In this study, we devised a biodegradable bilayered scaffold, comprising chondroitin sulfate (CS) hydrogel to regenerate chondral tissue and a porous pure zinc (Zn) scaffold for regeneration of the underlying bone as mechanical support for the cartilage layer. The photocured CS hydrogel possessed a compressive strength of 82 kPa, while the porous pure Zn scaffold exhibited a yield strength of 11 MPa and a stiffness of 0.8 GPa. Such mechanical properties are similar to values reported for cancellous bone. In vitro biological experiments demonstrated that the bilayered scaffold displayed favorable cytocompatibility and promoted chondrogenic and osteogenic differentiation of bone marrow stem cells. Upon implantation, the scaffold facilitated the simultaneous regeneration of bone and cartilage tissue in a porcine model, resulting in (i) a smoother cartilage surface, (ii) more hyaline-like cartilage, and (iii) a superior integration into the adjacent host tissue. Our bilayered scaffold exhibits significant potential for clinical application in osteochondral regeneration.

Nanofiber Composite Microchannel-Containing Injectable Hydrogels for Cartilage Tissue Regeneration.

Articular cartilage tissue is incapable of self-repair and therapies for cartilage defects are still lacking. Injectable hydrogels have drawn much attention in the field of cartilage regeneration. Herein, the novel design of nanofiber composite microchannel-containing hydrogels inspired by the tunnel-piled structure of subway tunnels is proposed. Based on the aldehydized polyethylene glycol/carboxymethyl chitosan (APA/CMCS) hydrogels, thermosensitive gelatin microrods (GMs) are used as a pore-forming agent, and coaxial electrospinning polylactic acid/gelatin fibers (PGFs) loaded with kartogenin (KGN) are used as a reinforcing agent and a drug delivery system to construct the nanofiber composite microchannel-containing injectable hydrogels (APA/CMCS/KGN@PGF/GM hydrogels). The in situ formation, micromorphology and porosity, swelling and degradation, mechanical properties, self-healing behavior, as well as drug release of the nanofiber composite microchannel-containing hydrogels are investigated. The hydrogel exhibits good self-healing ability, and the introduction of PGF nanofibers can significantly improve the mechanical properties. The drug delivery system can realize sustained release of KGN to match the process of cartilage repair. The microchannel structure effectively promotes bone marrow mesenchymal stem cell (BMSC) proliferation and ingrowth within the hydrogels. In vitro and animal experiments indicate that the APA/CMCS/KGN@PGF/GM hydrogels can enhance the chondrogenesis of BMSCs and promote neocartilage formation in the rabbit cartilage defect model.

Alginate Conjugation Increases Toughness in Auricular Chondrocyte Seeded Collagen Hydrogels.

Current auricular cartilage replacements for pediatric microtia fail to address the need for long-term integration and neocartilage formation. While collagen hydrogels have been successful in fostering neocartilage formation, the toughness and extensibility of these materials do not match that of native tissue. This study used the N-terminal functionalization of collagen with alginate oligomers to improve toughness and extensibility through metal-ion complexation. Alginate conjugation was confirmed via FTIR spectroscopy. The retention of native collagen fibrillar structure, thermal gelation, and helical conformation in functionalized gels was confirmed via scanning electron microscopy, oscillatory shear rheology, and circular dichroism spectroscopy, respectively. Alginate-calcium complexation enabled a more than two-fold increase in modulus and work density in functionalized collagen with the addition of 50 mM CaCl2, whereas unmodified collagen decreased in both modulus and work density with increasing calcium concentration. Additionally, the extensibility of alginate-functionalized collagen was increased at 25 and 50 mM CaCl2. Following 2-week culture with auricular chondrocytes, alginate-functionalization had no effect on the cytocompatibility of collagen gels, with no effects on cell density, and increased glycosaminoglycan deposition. Custom MATLAB video analysis was then used to quantify fracture toughness, which was more than 5-fold higher following culture in functionalized collagen and almost three-fold higher in unmodified collagen.

A Pilot Study of Decellularized Cartilage for Laryngotracheal Reconstruction in a Neonatal Pig Model.

Severe subglottic stenosis develops as a response to intubation in 1% of the >200,000 neonatal intensive care unit infants per year and may require laryngotracheal reconstruction (LTR) with autologous hyaline cartilage. Although effective, LTR is limited by comorbidities, severity of stenosis, and graft integration. In children, there is a significant incidence of restenosis requiring revision surgery. Tissue engineering has been proposed to develop alterative grafting options to improve outcomes and eliminate donor-site morbidity. Our objective is to engineer a decellularized, channel-laden xenogeneic cartilage graft, that we deployed in a proof-of-concept, neonatal porcine LTR model. Meniscal porcine cartilage was freeze-thawed and washed with pepsin/elastase to decellularize and create microchannels. A 6 × 10-mm decellularized cartilage graft was then implanted in 4 infant pigs in an anterior cricoid split. Airway patency and host response were monitored endoscopically until sacrifice at 12 weeks, when the construct phenotype, cricoid expansion, mechanics, and histomorphometry were evaluated. The selective digestion of meniscal components yielded decellularized cartilage with cell-size channels. After LTR with decellularized meniscus, neonatal pigs were monitored via periodic endoscopy observing re-epithelization, integration, and neocartilage formation. At 12 weeks, the graft appeared integrated and exhibited airway expansion of 4 mm in micro-CT and endoscopy. Micro-CT revealed a larger lumen compared with age-matched controls. Finally, histology showed significant neocartilage formation. Our neonatal porcine LTR model with a decellularized cartilage graft is a novel approach to tissue engineered pediatric LTR. This pilot study sets the stage for "off-the-shelf" graft procurement and future optimization of MEND for LTR. NA Laryngoscope, 134:807-814, 2024.

Regenerative Engineering of a Biphasic Patient-Fitted Temporomandibular Joint Condylar Prosthesis.

Regenerative medicine approaches to restore the mandibular condyle of the temporomandibular joint (TMJ) may fill an unmet patient need. In this study, a method to implant an acellular regenerative TMJ prosthesis was developed for orthotopic implantation in a pilot goat study. The scaffold incorporated a porous, polycaprolactone-hydroxyapatite (PCL-HAp, 20wt% HAp) 3D printed condyle with a cartilage-matrix-containing hydrogel. A series of material characterizations was used to determine the structure, fluid transport, and mechanical properties of 3D printed PCL-HAp. To promote marrow uptake for cell seeding, a scaffold pore size of 152 ± 68 μm resulted in a whole blood transport initial velocity of 3.7 ± 1.2 mm·s-1 transported to the full 1 cm height. The Young's modulus of PCL was increased by 67% with the addition of HAp, resulting in a stiffness of 269 ± 20 MPa for etched PCL-HAp. In addition, the bending modulus increased by 2.06-fold with the addition of HAp to 470 MPa for PCL-HAp. The prosthesis design with an integrated hydrogel was compared with unoperated contralateral control and no-hydrogel group in a goat model for 6 months. A guide was used to make the condylectomy cut, and the TMJ disc was preserved. MicroCT assessment of bone suggested variable tissue responses with some regions of bone growth and loss, although more loss may have been exhibited by the hydrogel group than the no-hydrogel group. A benchtop load transmission test suggested that the prosthesis was not shielding load to the underlying bone. Although variable, signs of neocartilage formation were exhibited by Alcian blue and collagen II staining on the anterior, functional surface of the condyle. Overall, this study demonstrated signs of functional TMJ restoration with an acellular prosthesis. There were apparent limitations to continuous, reproducible bone formation, and stratified zonal cartilage regeneration. Future work may refine the prosthesis design for a regenerative TMJ prosthesis amenable to clinical translation.

Evaluation of surgical fixation methods for the implantation of melt electrowriting-reinforced hyaluronic acid hydrogel composites in porcine cartilage defects.

The surgical repair of articular cartilage remains an ongoing challenge in orthopedics. Tissue engineering is a promising approach to treat cartilage defects; however, scaffolds must (i) possess the requisite material properties to support neocartilage formation, (ii) exhibit sufficient mechanical integrity for handling during implantation, and (iii) be reliably fixed within cartilage defects during surgery. In this study, we demonstrate the reinforcement of soft norbornene-modified hyaluronic acid (NorHA) hydrogels via the melt electrowriting (MEW) of polycaprolactone to fabricate composite scaffolds that support encapsulated porcine mesenchymal stromal cell (pMSC, three donors) chondrogenesis and cartilage formation and exhibit mechanical properties suitable for handling during implantation. Thereafter, acellular MEW-NorHA composites or MEW-NorHA composites with encapsulated pMSCs and precultured for 28 days were implanted in full-thickness cartilage defects in porcine knees using either bioresorbable pins or fibrin glue to assess surgical fixation methods. Fixation of composites with either biodegradable pins or fibrin glue ensured implant retention in most cases (80%); however, defects treated with pinned composites exhibited more subchondral bone remodeling and inferior cartilage repair, as evidenced by micro-computed tomography (micro-CT) and safranin O/fast green staining, respectively, when compared to defects treated with glued composites. Interestingly, no differences in repair tissue were observed between acellular and cellularized implants. Additional work is required to assess the full potential of these scaffolds for cartilage repair. However, these results suggest that future approaches for cartilage repair with MEW-reinforced hydrogels should be carefully evaluated with regard to their fixation approach for construct retention and surrounding cartilage tissue damage.

Cartilage Lacuna-Inspired Microcarriers Drive Hyaline Neocartilage Regeneration.

Cartilage equivalents from hydrogels containing chondrocytes exhibit excellent potential in hyaline cartilage regeneration, yet current approaches have limited success at reconstituting the architecture to culture nondifferentiated chondrocytes in vitro. In this study, specially designed lacunar hyaluronic acid microcarriers (LHAMCs) with mechanotransductive conditions that rapidly form stable hyaluronic acid (HA) N-hydroxy succinimide ester (NHS-ester) are reported. Specifically, carboxyl-functionalized HA is linked to collagen type I via amide-crosslinking, and gas foaming produced by ammonium bicarbonate forms concave surface of the microcarriers. The temporal 3D culture of chondrocytes on LHAMCs uniquely remodels the extracellular matrix to induce hyaline cartilaginous microtissue regeneration and prevents an anaerobic-to-aerobic metabolism transition in response to the geometric constraints. Furthermore, by inhibiting the canonical Wnt pathway, LHAMCs prevent β-catenin translocation to the nucleus, repressing chondrocyte dedifferentiation. Additionally, the subcutaneous implantation model indicates that LHAMCs display favorable cytocompatibility and drive robust hyaline chondrocyte-derived neocartilage formation. These findings reveal a novel strategy for regulating chondrocyte dedifferentiation. The current study paves the way for a better understanding of geometrical insight clues into mechanotransduction interaction in regulating cell fate, opening new avenues for advancing tissue engineering.

Neo-cartilage formation using human nondegenerate versus osteoarthritic chondrocyte-derived cartilage organoids in a viscoelastic hydrogel.

Current regenerative cartilage therapies are associated with several drawbacks such as dedifferentiation of chondrocytes during expansion and the formation of fibrocartilage. Optimized chondrocyte expansion and tissue formation could lead to better clinical results of these therapies. In this study, a novel chondrocyte suspension expansion protocol that includes the addition of porcine notochordal cell-derived matrixwas used to self-assemble human chondrocytes from osteoarthritic (OA) and nondegenerate (ND) origin into cartilage organoids containing collagen type II and proteoglycans. Proliferation rate and viability were similar for OA and ND chondrocytes and organoids formed had a similar histologic appearance and gene expression profile. Organoids were then encapsulated in viscoelastic alginate hydrogels to form larger tissues. Chondrocytes on the outer bounds of the organoids produced a proteoglycan-rich matrix to bridge the space between organoids. In hydrogels containing ND organoids some collagen type I was observed between the organoids. Surrounding the bulk of organoids in the center of the gels, in both OA and ND gels a continuous tissue containing cells, proteoglycans and collagen type II had been produced. No difference was observed in sulphated glycosaminoglycan and hydroxyproline content between gels containing organoids from OA or ND origin after 28 days. It was concluded that OA chondrocytes, which can be harvested from leftover surgery tissue, perform similar to ND chondrocytes in terms of human cartilage organoid formation and matrix production in alginate gels. This opens possibilities for their potential to serve as a platform for cartilage regeneration but also as an in vitro model to study pathways, pathology, or drug development.

Enzymatic isolation of articular chondrons: is it much different than that of chondrocytes?

In native articular cartilage, chondrocytes are completely capsulated by a pericellular matrix (PCM), together called the chondron. Due to its unique properties (w.r.t. territorial matrix) and importance in mechanotransduction, the PCM and chondron may be important in regenerative strategies. The current gold standard for the isolation of chondrons from cartilage dates from 1997. Although previous research already showed the low cell yield and the heterogeneity of the isolated populations, their compositions and properties have never been thoroughly characterized. This study aimed to compare enzymatic isolation methods for chondrocytes and chondrons and characterize the isolation efficiency and quality of the PCM. Bovine articular cartilage was digested according to the 5-hour gold standard chondron isolation method (0.3% dispase + 0.2% collagenase II), an overnight chondron isolation (0.15% dispase + 0.1% collagenase II), and an overnight chondrocyte isolation (0.15% collagenase II + 0.01% hyaluronidase). Type VI collagen staining, fluorescence-activated cell sorting (FACS) analysis, specific cell sorting and immunohistochemistry were performed using a type VI collagen staining, to study their isolation efficiency and quality of the PCM. These analyses showed a heterogeneous mixture of chondrocytes and chondrons for all three methods. Although the 5-hour chondron isolation resulted in the highest percentage of chondrons, the cell yield was significantly lower compared to the other isolation methods. FACS, based on the type VI collagen staining, successfully sorted the three identified cell populations. To maximize chondron yield and homogeneity, the overnight chondron enzymatic digestion method should be combined with type VI collagen staining and specific cell sorting.

From regeneration to osteoarthritis in the knee joint: The role shift of cartilage-derived progenitor cells.

A mount of growing evidence has proven that cartilage-derived progenitor cells (CPCs) harbor strong proliferation, migration, andmultiple differentiation potentials over the past 2 decades. CPCs in the stage of immature tissue play an important role in cartilage development process and injured cartilage repair in the young and active people. However, during maturation and aging, cartilage defects cannot be completely repaired by CPCs in vivo. Recently, tissue engineering has revealed that repaired cartilage defects with sufficient stem cell resources under good condition and bioactive scaffolds in vitro and in vivo. Chronic inflammation in the knee joint limit the proliferation and chondrogenesis abilities of CPCs, which further hampered cartilage healing and regeneration. Neocartilage formation was observed in the varus deformity of osteoarthritis (OA) patients treated with offloading technologies, which raises the possibility that organisms could rebuild cartilage structures spontaneously. In addition, nutritionmetabolismdysregulation, including glucose and free fatty acid dysregulation, could influence both chondrogenesis and cartilage formation. There are a few reviews about the advantages of CPCs for cartilage repair, but few focused on the reasons why CPCs could not repair the cartilage as they do in immature status. A wide spectrum of CPCs was generated by different techniques and exhibited substantial differences. We recently reported that CPCs maybe are as internal inflammation sources during cartilage inflammaging. In this review, we further streamlined the changes of CPCs from immature development to maturation and from healthy status to OA advancement. The key words including "cartilage derived stem cells", "cartilage progenitor cells", "chondroprogenitor cells", "chondroprogenitors" were set for latest literature searching in PubMed and Web of Science. The articles were then screened through titles, abstracts, and the full texts in sequence. The internal environment including long-term inflammation, extendedmechanical loading, and nutritional elements intake and external deleterious factors were summarized. Taken together, these results provide a comprehensive understanding of the underlying mechanism of CPC proliferation and differentiation during development, maturation, aging, injury, and cartilage regeneration in vivo.

Proteomic, mechanical, and biochemical development of tissue-engineered neocartilage

BackgroundThe self-assembling process of cartilage tissue engineering is a promising technique to heal cartilage defects, preventing osteoarthritic changes. Given that chondrocytes dedifferentiate when expanded, it is not known if cellular expansion affects the development of self-assembled neocartilage. The objective of this study was to use proteomic, mechanical, and biochemical analyses to quantitatively investigate the development of self-assembled neocartilage derived from passaged, rejuvenated costal chondrocytes.MethodsYucatan minipig costal chondrocytes were used to create self-assembled neocartilage constructs. After 1, 4, 7, 14, 28, 56, or 84 days of self-assembly, constructs were analyzed through a variety of histological, biomechanical, biochemical, and proteomic techniques.ResultsIt was found that temporal trends in neocartilage formation are similar to those seen in native hyaline articular cartilage development. For example, between days 7 and 84 of culture, tensile Young’s modulus increased 4.4-times, total collagen increased 2.7-times, DNA content decreased 69.3%, collagen type II increased 1.5-times, and aggrecan dropped 55.3%, mirroring trends shown in native knee cartilage. Importantly, collagen type X, which is associated with cartilage calcification, remained at low levels (≤ 0.05%) at all neocartilage developmental time points, similar to knee cartilage (< 0.01%) and unlike donor rib cartilage (0.98%). ConclusionsIn this work, bottom-up proteomics, a powerful tool to interrogate tissue composition, was used for the first time to quantify and compare the proteome of a developing engineered tissue to a recipient tissue. Furthermore, it was shown that self-assembled, costal chondrocyte-derived neocartilage is suitable for a non-homologous approach in the knee.

Functionalized Electrospun Scaffold-Human-Muscle-Derived Stem Cell Construct Promotes In Vivo Neocartilage Formation.

Polycaprolactone (PCL) is a non-cytotoxic, completely biodegradable biomaterial, ideal for cartilage tissue engineering. Despite drawbacks such as low hydrophilicity and lack of functional groups necessary for incorporating growth factors, it provides a proper environment for different cells, including stem cells. In our study, we aimed to improve properties of scaffolds for better cell adherence and cartilage regeneration. Thus, electrospun PCL–scaffolds were functionalized with ozone and loaded with TGF-β3. Together, human-muscle-derived stem cells (hMDSCs) were isolated and assessed for their phenotype and potential to differentiate into specific lineages. Then, hMDSCs were seeded on ozonated (O) and non-ozonated (“naïve” (NO)) scaffolds with or without protein and submitted for in vitro and in vivo experiments. In vitro studies showed that hMDSC and control cells (human chondrocyte) could be tracked for at least 14 days. We observed better proliferation of hMDSCs in O scaffolds compared to NO scaffolds from day 7 to day 28. Protein analysis revealed slightly higher expression of type II collagen (Coll2) on O scaffolds compared to NO on days 21 and 28. We detected more pronounced formation of glycosaminoglycans in the O scaffolds containing TGF-β3 and hMDSC compared to NO and scaffolds without TGF-β3 in in vivo animal experiments. Coll2-positive extracellular matrix was observed within O and NO scaffolds containing TGF-β3 and hMDSC for up to 8 weeks after implantation. These findings suggest that ozone-treated, TGF-β3-loaded scaffold with hMDSC is a promising tool in neocartilage formation.

A transcriptome sequencing study on the effect of macro-pores in hydrogel scaffolds on global gene expression of laden human cartilage chondrocytes

The macro-porous hydrogel scaffolds can not only enhance the proliferation of laden chondrocytes but also favor the deposition of hyaline cartilaginous extracellular matrix, however, the underlying molecular mechanism is still unclear. Herein, the global gene expression of human cartilage chondrocytes (HCCs) encapsulated in traditional hydrogel (Gel) constructs and micro-cavitary gel (MCG) constructs are investigated by using high-throughput RNA sequencing (RNA-seq). The differentially expressed genes (DEGs) between the HCCs cultured in Gel and MCG constructs have been identified via bioinformatics analysis. Significantly, the DEGs that promote cell proliferation (e.g. POSTN, MKI67, KIF20A) or neo-cartilage formation (e.g. COL2, ASPN, COMP, FMOD, FN1), are more highly expressed in MCG constructs than in Gel constructs, while the expressions of the DEGs associated with chondrocyte hypertrophy (e.g. EGR1, IBSP) are upregulated in Gel constructs. The expression of representative DEGs is verified at both mRNA and protein levels. Besides, cellular viability and morphology as well as the enriched signaling pathway of DEGs are studied in detail. These results of this work may provide data for functional tissue engineering of cartilage.

Standardised quantitative ultrasound imaging approach for the contact-less three-dimensional analysis of neocartilage formation in hydrogel-based bioscaffolds

In this work we present a standardised quantitative ultrasound imaging (SQUI) approach for the non-destructive three-dimensional imaging and quantification of cartilage formation in hydrogel based bioscaffolds. The standardised concept involves the processing of ultrasound backscatter data with respect to an acellular phantom in combination with the compensation of sound speed mismatch diffraction effects between the bioscaffold and the phantom. As a proof-of-concept, the SQUI approach was tested on a variety of bioscaffolds with varying degree of neocartilage formation. These were composed of Gelatine Methacryloyl (GelMA) hydrogels laden with human adipose-derived stem cells (hADSCs). These were cultured under chondrogenic stimulation following a previously established protocol, where the degree of the neocartilage formation was modulated using different GelMA network densities (6, 8, 10 % w/v) and culture time (0, 14, 28 days). Using the SQUI approach we were able to detect marked acoustic and morphological changes occurring in the bioscaffolds a result of their different chondrogenic outcome. We defined an acoustic neocartilage indicator, the sonomarker, for the selective imaging and quantification of neocartilage formation. The sonomarker, of backscatter intensity logIBC -2.4, was found to correlate with data obtained via standard destructive bioassays. The ultrasonic evaluation of human specimens confirmed the sonomarker as a relevant intensity, although it was found to shift to higher intensity values in proportion to the cartilage condition as inferred from sound speed measurements. This study demonstrates the potential of the SQUI approach for the realization of non-destructive analysis of cartilage regeneration over-time. Statement of significanceAs tissue engineering strategies for neocartilage regeneration evolve towards clinical implementation, alternative characterisation approaches that allow the non-destructive monitoring of extracellular matrix formation in implantable hydrogel based bioscaffolds are needed. In this work we present an innovative standardized quantitative ultrasound imaging (SQUI) approach that allows the non-destructive, volumetric, and quantitative evaluation of neocartilage formation in hydrogel based bioscaffolds. The standardised concept aims to provide a robust approach that accounts for the dynamic changes occurring during the conversion from a cellular bioscaffold towards the formation of a neocartilage construct. We believe that the SQUI approach will be of great benefit for the evaluation of constructs developing neocartilage, not only for in-vitro applications but also potentially applicable to in-vivo applications.

Potential of Using Infrapatellar-Fat-Pad-Derived Mesenchymal Stem Cells for Therapy in Degenerative Arthritis: Chondrogenesis, Exosomes, and Transcription Regulation.

Infrapatellar fat pad–derived mesenchymal stem cells (IPFP-MSCs) are a type of adipose-derived stem cell (ADSC). They potentially contribute to cartilage regeneration and modulation of the immune microenvironment in patients with osteoarthritis (OA). The ability of IPFP-MSCs to increase chondrogenic capacity has been reported to be greater, less age dependent, and less affected by inflammatory changes than that of other MSCs. Transcription-regulatory factors strictly regulate the cartilage differentiation of MSCs. However, few studies have explored the effect of transcriptional factors on IPFP-MSC-based neocartilage formation, cartilage engineering, and tissue functionality during and after chondrogenesis. Instead of intact MSCs, MSC-derived extracellular vesicles could be used for the treatment of OA. Furthermore, exosomes are increasingly being considered the principal therapeutic agent in MSC secretions that is responsible for the regenerative and immunomodulatory functions of MSCs in cartilage repair. The present study provides an overview of advancements in enhancement strategies for IPFP-MSC chondrogenic differentiation, including the effects of transcriptional factors, the modulation of released exosomes, delivery mechanisms for MSCs, and ethical and regulatory points concerning the development of MSC products. This review will contribute to the understanding of the IPFP-MSC chondrogenic differentiation process and enable the improvement of IPFP-MSC-based cartilage tissue engineering.