Introduction

The original hypothesis and expectation when the cell therapy for cartilage regeneration first started was that the implanted cells would survive, engraft to chondral defects, and differentiated to articular chondrocyte that would subsequently produce extracellular matrix (ECM). The cell therapy would thus achieve structural modification of damaged joint by regeneration of AC, which might also eventually supplant conventional treatments for OA. Therefore, early experimental studies for MSC-based cartilage regeneration focused on the chondrogenic induction from MSCs. Numerous studies were performed to induce chondrogenesis from MSC by the appropriate combination of growth factors or transforming cells by transfer of chondrogenic genes. Other studies also investigated the application of biomechanical stimuli to enhance chondrogenic differentiation from MSCs.10-11 As one distinctive shortcoming of MSCs as chondrogenic cell source was early induction of hypertrophic markers such as type X collagen, great efforts were made to devise ways to suppress hypertrophy in MSC chondrogenesis.

Content

The tracking of administered cell is essential for understanding the mode of action in a stem cell therapy. Whilst engraftment and chondrogenic differentiation was purposed in the beginning, on the other hand, it became apparent that IA-administered cells survive only transiently and undergo rapid cell death as also reported from stem cell implantation in other tissues such as myocardium. Most IA-administered stem cells undergo rapid cell death, surviving from 3 days to several weeks depending on the mode of administration and the IA environment. Paracrine factors that are released before undergoing apoptosis possess predominantly immunosuppressive and anti-inflammatory actions rather than chondrogenic effects. Rapid death of implanted cells is not limited to MSCs. Chondrocytes also undergo rapid clearance when injected into joints within 2 weeks. Interestingly, stem cells survived longer when focally implanted rather than injected. Rapid death of administered cells in vivo makes the efforts for the chondrogenic differentiation or hypertrophy inhibition in vitro quite meaningless. Instead, the focus of research then can move to prolongation of survival of implanted cells that could exert prolonged paracrine effects and/or engraftment with chondrogenic differentiation. For example, adipose stem cells (ASCs) in spheroid form survive longer post-IA injection than do ASCs that are injected in a free single cell suspension. These findings suggest that a sort of communication/interaction between cells can promote IA cell survival. Also, our preliminary experiments demonstrated that when ASCs were immobilized on the focal chondral defect using a strong bioadhesive (mussel adhesive protein) showed longer-term survival than those fixed using weak adhesive such as fibrin glue. These results indicate that stem cells can survive longer when forced to stay at the site of implantation. While it is not yet proven that these preliminary findings of prolonged cell survival may be translated into tangible difference in clinical application, the concept deserves further inquiry and investigation.

References

1. Ko JY, Park JW, Kim J, Im GI. Characterization of adipose-derived stromal/stem cell spheroids versus single-cell suspension in cell survival and arrest of osteoarthritis progression. J Biomed Mater Res A. 2021 Jun;109(6):869-87

2. Im GI. Current status of regenerative medicine in osteoarthritis. Bone Joint Res. 2021 Feb;10(2):134-136

3. Im GI, Kim TK. Regenerative Therapy for Osteoarthritis: A Perspective.Int J Stem Cells. 2020 Jul 30;13(2):177-181

Acknowledgments

This research was supported by a grant of the National Research Foundation of
Korea (NRF-2020R1A2C2008266).

Introduction

The regeneration of cartilage tissue in vitro is often dependent on a scaffold material with the biomimetic properties. Ideally the scaffold should mimic the properties of native extracellular matrix: it should allow the cells to divide, secrete phenotypic matrix molecules and also to remodeling the matrix in response to microinjuries. Hydrogels are often used as a scaffold material due to their high water content and inherent biocompatibility. As a polymer backbone, hyaluronan is one of the most common materials used as it is a component of the native cartilage extracellular matrix and has multiple groups for chemical functionalization. In this study we have explored the use of a hyaluronic acid hydrogel (HATG) in the presence and absence of alginate. The hydrogel undergoes crosslinking in the presence of activated Factor XIII. We studied the maturation of these chondrocyte-containing constructs using mechanical testing (compression and bioindentation measurements) and histological evaluations.

In order to reproduce more complex structures which could be used to treat patient specific defects, it is possible to render HA-TG processible in a bioprinter. To acheive this, nanofibers were added to the HATG/HATG-alg solutions and the rheological properties of the bioink measured. We able to bioprint HATG into unique shapes and investigate their properties in vitro and in vivo in subcutaneous nude rat models . In future work, we will also explore the challenges of cultivating large tissue and potential benefits of bioreactors and gene editing of the cells to improve the mechanical stability of the printed cartilage.

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Human chondrocytes were isolated from biopsies taken with informed patient consent. Chondrocytes were expanded to passage 3 in media containing fetal bovine serum, TGFb3 and FGF2. Cells were trypsinized and encapsulated in the HATG or HATG-Alg materials at a cell density of 15mio cells/ml and the constructs were cultured for up to 9 weeks in TGFb3 containing DMEM. The stability of the cartilage constructs in vivo was explored by transplanted the discs in a subcutaneous nude rat model.

The chondrocytes in HATG and HATG-Alg discs showed increase in modulus approaching 1 MPa after 9 week in vitro culture, with maturation of the cartilage more efficient in HATG-Alg. By day 21 of culture, the chondrocyte viability in both HATG and HATG-alg approached over 90%. Safranin O staining likewise increased over time in culture, approaching the staining intensity of native auricular cartilage. Immunohistochemistry analyses revealed copious deposition of both collagen II and collagen I types as well as elastin staining. Large, bioprinted HATG and HATG-alg constructs developed slower and showed poor structural stability in vivo, perhaps due to inadequate diffusion of nutrients.

In summary, HATG and in particular HATG-Alg provide a 3D environment highly conducive to the formation of robust cartilage tissue in vitro. Methods to improve the in vivo tissue stability of large constructs are being explored and include the addition of porosity in the scaffold, enhanced crosslinking of the scaffold, the use of bioreactors and increasing the stability of the cells through gene editing.

References

Fisch et al, Advanced Functional Materials, https://doi.org/10.1002/adfm.202008261

Acknowledgments

Funding from the Swiss National Science Foundation CRSII5_173868 is gratefully acknowledged.

Introduction

Biofabrication offers a range of techniques and methods to build constructs with desired shapes and internal gradients of composition and cell types. Although achieving desired shapes and composition is fundamental, tissue properties also depend on the internal architecture at the microscopic level. For example, cartilage mechanical and biological properties critically depend on the orientation of collagen (col) fibrils and their zonal organization. Although biofabrication methods to control microscopic architecture are relatively mature, they often require sophisticated techniques and are low in throughput; additionally, their integration with the most accessible biofabrication methods is complex. Here, we present two techniques to control the spatial arrangement of collagen fibrils and of porosity within composite constructs based on hyaluronic acid (HA) and col. Col fibrils orientation was controlled via extrusion-based 3D printing after embedding within a continuous viscoelastic matrix based on crosslinked HA. Cell-free and cell laden constructs were prepared and characterized to determine the influence of this controlled microscopic anisotropy on cell behavior. Chondrogenic properties of the composites were assessed for a range of HA/col compositions by evaluating human bone marrow derived mesenchymal stromal cell (hMSC) differentiation to the chondrogenic lineage.

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Materials and Methods

The tyramine derivative of HA (THA) was prepared by functionalizing the carboxy groups of HA via amide bond formation. THA was combined with soluble acidic col at the following THA:col concentrations: 12.5:2.5; 12.5:1.7; 16.7:2.5; 12.5:1.7 mg/ml. The mixture was treated with hydrogen peroxide 18 to 22 ppm in presence of horseradish peroxidase [1] for enzymatic crosslinking of THA, and at the same time pH was shifted to neutrality at 37°C for inducing col fibrillogenesis. Shear thinning and viscoelastic properties of THA, col and composites were characterized via rheometry (AntonPaar MCR302) measuring the viscosity as a function of the shear rate, with an amplitude sweep and with an elastic recovery test performed alternating intervals of high and low strain to assess the kinetic recovery after deformation. The THA-col composite was printed (3D Discovery, RegenHU) controlling col fiber alignment with the shear forces acting during extrusion. Col orientation was visualized via Second Harmonic Generation (SHG) and immunostaining/confocal microscopy. Images were processed with image J (NIH) to quantify fiber orientation. hMSCs (passage 3) were preformed into aggregates by seeding into microwell plates (Aggrewell 400, Stemcell Technology) according to the manufacturer’s protocol using chondrogenic media (DMEM HG, 50 µg/ml ascorbic acid 2-phosphate, 100 nM dexamethasone, 1% v/v ITS premix, 1% v/v non-essential amino acids (MEM NEAA, Gibco), 10 ng/ml transforming growth factor beta (TGF-β1, Fitzgerald), 1% v/v Pen-Strep and cultured for 3 days. The obtained aggregates contained approximately 250 hMSCs and were embedded within the bioink to evaluate cell migration and orientation, assessed by F-actin and DAPI staining (Fig 1 D). In vitro chondrogenic behavior of hMSC embedded in casted THA-col (chondrogenic media containing 10 ng/ml TGF-β1) was evaluated via histology, gene expression (COL1A1, COL2A1, COL10A1, RunX2, SOX9, Aggrecan ACAN) and proteoglycan quantification. Chondrogenic differentiation was compared to hMSC pellets as a positive control.

Results

The gel nature of collagen and crosslinked HA prevents efficacious mixing. To obtain a homogeneous dispersion of col fibrils within the crosslinked HA gel, a method was developed starting from soluble acidic col and an non-crosslinked THA [2]. The liquid nature of these precursors allowed thorough mixing, followed by simultaneous col fibril formation and THA crosslinking avoiding mutual interference to achieve uniform distribution of col fibers within the HA-based viscoelastic matrix as seen in Fig 1 B.

Shear-induced fiber alignment along the printing direction was shown via immunofluorescence, SHG (Figure 1 A, B) and by immunohistochemistry (not shown). As expected, fiber alignment increased for decreasing nozzle diameter (Fig 1 C, D). The shear thinning and elastic recovery of THA [1] were preserved in the THA-col composite at the investigated mixing ratios. THA-col showed 2-fold increase in storage modulus compared to THA.

The presence of col fibers had an evident impact on cell attachment and migration. Actin filament staining showed cytoskeleton alignment along the fiber orientation, which in turn was determined by the printing direction (Fig 1C). Production of proteoglycan rich extracellular matrix during chondrogenesis was observed after 3 weeks of hMSC in vitro culture embedded in THA-col (figure 2B). At 3 weeks, chondrogenic associated genes COL2A1 (>100-fold), ACAN (>100-fold) and SOX9 were upregulated in all samples. Compared to hMSC pellets, hypertrophic markers (COL1A1, COL10A1, RunX2) were less upregulated in the THA-col hydrogels with higher ratio of SOX9/RunX2 in both THA-col samples (~10-fold) compared to no change in the pellet group (~1-fold).

Discussion

In this work, we introduce a method to 3D bioprint col with controlled orientation embedded within a viscoelastic HA matrix. Introducing control over the microscopic architecture of matrix components is key to recapitulate complex tissue structure and morphology. One potential limitation of the approach here presented is the use of the shear forces for alignment, limiting the space of design possibilities. Also, it remains to be determined how this controlled orientation influences matrix deposition. Overall, the possibility of printing matrix components with control over microscopic alignment brings biofabrication one step closer to capturing the complexity in animal tissues.

References

[1] Petta D C et al. Biofabrication. 2018 Sep 25;10(4):044104; PMID: 30188324

[2] Schwab et al. Mater Today Bio. 2020 Jun 1;7:100058; PMID: 3261318

Acknowledgments

This work is part of the osteochondral defect collaborative research program supported by the AO foundation. The Graubünden Innovationsstiftung is acknowledged for its financial support.

Introduction

Biomaterials are often used to support the 3D environment of the implanted tissue as well as to reinforce adhesion to the osteochondral lesion base and surrounding tissue. However, there have been few studies to clearly elucidate the significance of the presence of mesenchymal stromal cells (MSCs) within the implanted biomaterials. In discussing whether cell-based or cell-free strategy to be used, it is important to address to bio- and mechanical function of the tested materials, however we have not had appropriate experimental model to test the significance of living MSCs within the biomaterials in the repair and remodeling of osteochondral lesions.

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Here we introduce a model of osteochondral repair using iPS cell-derived cartilaginous spheroids and synovial MSCs-derived tissue engineered construct whose matrix are natural matrix produced by the MSCs. Human iPSC-derived cartilaginous particles (iPSC-CP) developed via sequencial in vitro differentiation are a spherical body of hyaline cartilage-like tissue (1), which could be implanted to the osteochondral defect to repair and regenerate the lesion. However, due to its relatively low adhesive properties, the spheroid was wrapped by MSC-based tissue-engineered constructs (TEC) (2) to secure adhesion to the lesion base and surrounding osteochondral tissue. As the control, Freeze-dried TEC was used to wrap the spheroid. Regardless of freeze-dried preparation, wrapped spheroids attained secure defect filling with good integration to adjacent tissue in a rat osteochondral injury model over a 24-week post-implantation period. Conversely, the outcome of spheroid implantation was totally different between the TEC and Freeze-dried TEC group. The presence of living MSCs in the hybrid implants (TEC group) contributed to accomplish biphasic osteochondral repair. Defects reconstituted with a freeze-dried TEC initially maintained the original matrix of the spheroid but hereafter did not support osteochondral repair. Thus, it has been revealed that living MSCs have crucial function to induce proper biphasic osteochondral repair potentially by facilitating the communication with recipient cells.

Although this study use only one specific type of cell (synovial mesenchymal stromal cells) and matrix developed by the synovial MSCs, the present study suggests the presence of MSCs within natural matrix significantly determine the out come of tissue repair and remodelling. The use of iPC-derived chondral pheroid-based osteochondral repair model could be a good model to test the significant relation between pupulating MSCs and biomaterials. Further studies are required to test the phenomenon observed in the present study is applicable to other cells and biomaterials.

References

1. Generation of scaffoldless hyaline cartilaginous tissue from human iPSCs. Yamashita A, Morioka M, Yahara Y, Okada M, Kobayashi T, Kuriyama S, Matsuda S, Tsumaki N. Stem Cell Reports. 2015 Mar 10;4(3):404-18.

2. Cartilage repair using an in vitro generated scaffold-free tissue-engineered construct derived from porcine synovial mesenchymal stem cells. Ando W, Tateishi K, Hart DA, Katakai D, Tanaka Y, Nakata K, Hashimoto J, Fujie H, Shino K, Yoshikawa H, Nakamura N. Biomaterials. 2007 Dec;28(36):5462-70.

Acknowledgments

This study was supported by the grant for Research Center Network for Realization of Regenerative Medicine, Japan Agency for Medical Research and Development.

I appreciate Dr. N. Tsumaki for providing iPS cell-derived spheroids and for valuabke discussions.