a-proinflammatory-degenerative-organ-culture-model-to-simulate-early

A Proinflammatory, Degenerative Organ Culture Model to Simulate Early-Stage Intervertebral Disc Disease.

Symptomatic intervertebral disc (IVD) degeneration (IDD) is a major socioeconomic burden and is characterized by inflammation and tissue degradation. Due ...

AO Research Institute Davos, Davos, Switzerland

A cartilage bioreactor has been designed that is intended to approximate the kinematics of natural joints and allows for functional cartilage tissue engineering studies. In particular, interface motion can be generated by oscillation of a ball over the surface of a construct. The present study investigated the specific effect of applied articular motion on the gene expression of chondrocytes cultured in 3D scaffolds, with a particular emphasis on different superficial zone protein (SZP)/lubricin transcripts. Cylindrical porous polyurethane scaffolds were seeded with bovine articular chondrocytes and subjected to dynamic compression, with or without articulation against a ceramic hip ball. Articular motion markedly up-regulated the mRNA expression of the four previously described and two newly identified SZP/lubricin isoforms and of cartilage oligomeric matrix protein (COMP), and, to a lesser extent, aggrecan, type II collagen and TIMPs, while axial compression alone had no effect on the chondrocytes' gene expression levels. These results demonstrate the beneficial effect of articular motion not only for stimulation of important lubricating molecules, but also for the preservation of the chondrocytic phenotype.

Chondrocyte gene expression under applied surface motion.

This study investigated the effect of unidirectional and multidirectional motion patterns on gene expression and molecule release of chondrocyte-seeded 3D scaffolds. Resorbable porous polyurethane scaffolds were seeded with bovine articular chondrocytes and exposed to dynamic compression, applied with a ceramic hip ball, alone (group 1), with superimposed rotation of the scaffold around its cylindrical axis (group 2), oscillation of the ball over the scaffold surface (group 3), or oscillation of ball and scaffold in phase difference (group 4). Compared with group 1, the proteoglycan 4 (PRG4) and cartilage oligomeric matrix protein (COMP) mRNA expression levels were markedly increased by ball oscillation (groups 3 and 4). Furthermore, the collagen type II mRNA expression was enhanced in the groups 3 and 4, while the aggrecan and tissue inhibitor of metalloproteinase-3 (TIMP-3) mRNA expression levels were upregulated by multidirectional articular motion (group 4). Ball oscillation (groups 3 and 4) also increased the release of PRG4, COMP, and hyaluronan (HA) into the culture media. This indicates that the applied stimuli can contribute to the maintenance of the chondrocytic phenotype of the cells. The mechanical effects causing cell stimulation by applied surface motion might be related to fluid film buildup and/or frictional shear at the scaffold-ball interface. It is suggested that the oscillating ball drags the fluid into the joint space, thereby causing biophysical effects similar to those of fluid flow.

Effects of simple and complex motion patterns on gene expression of chondrocytes seeded in 3D scaffolds.

Chondrocytes represent the most important cell source for engineering of cartilaginous tissues. Depending on the tissue type and the localization within the tissue, these cells may behave differently. Numerous studies have been done to compare articular, nasal, auricular, and costal chondrocytes in order to evaluate differences between knee and ankle joint cartilage and to investigate topographical variations within an articular joint. Moreover, the zonal structure of articular cartilage needs to be considered because it leads to phenotypical differences between chondrocytes of the superficial and the deeper zones. Several studies indicate, however, that even differentiated chondrocytes demonstrate a certain plasticity and strive to adapt their phenotypes to a new mechanical and biochemical environment. The aim of this review is to report on similarities and differences of chondrocytes from different tissues, zones, and topographical locations. In particular, an overview of recent results from comparative studies is presented, and possible consequences for the design of tissue engineering models are discussed.

[Chondrocytes - one cell type, different subpopulations : characteristics and behavior of different types of chondrocytes and implications for tissue engineering applications].

Functional cartilage tissue engineering aims to generate grafts with a functional surface, similar to that of authentic cartilage. Bioreactors that stimulate cell-scaffold constructs by simulating natural joint movements hold great potential to generate cartilage with adequate surface properties. In this study two methods based on atomic force microscopy (AFM) were applied to obtain information about the quality of engineered graft surfaces. For better understanding of the molecule-function relationships, AFM was complemented with immunohistochemistry.

Sliding motion modulates stiffness and friction coefficient at the surface of tissue engineered cartilage.

Intervertebral disc (IVD) degeneration is the leading cause of low back pain and disability in the active population. Transplantation of mesenchymal stem cells (MSCs) in a hydrogel carrier can induce regenerative effects in degenerated IVDs. Moreover, it was found that degenerative discs release chemoattractants effective in MSC recruitment. Based on these findings, we hypothesized that an injectable hydrogel that can enhance the number of migrated MSCs in the IVD and provide a suitable matrix for their survival and differentiation would be ideal. The purpose of this study was to evaluate the potential of a thermoreversible hyaluronan-poly(N-isopropylacrylamide) (HAP) hydrogel as chemoattractant delivery system to recruit human MSCs in degenerative IVDs. The results demonstrate that HAP hydrogels containing stromal cell derived factor-1 (SDF-1) significantly increased the number of MSCs migrating into nucleotomized discs compared with discs treated with only HAP or SDF-1 in solution. HAP hydrogels releasing SDF-1 enhanced both the number of recruited cells and their migration distance in the IVD tissue. Furthermore, this phenomenon was dependent on MSC donor age. In conclusion, HAP SDF-1 is effective for the recruitment of stem cells in the IVD, thus opening new possibilities for the development of regenerative therapies based on endogenous cell migration. 

The effect of hyaluronan-based delivery of stromal cell-derived factor-1 on the recruitment of MSCs in degenerating intervertebral discs.

The healthy intervertebral disc (IVD) fulfils the essential function of load absorption, while maintaining multi-axial flexibility of the spine. The interrelated tissues of the IVD, the annulus fibrosus, the nucleus pulposus, and the cartilaginous endplate, are characterised by their specific niche, implying avascularity, hypoxia, acidic environment, low nutrition, and low cellularity. Anabolic and catabolic factors balance a slow physiological turnover of extracellular matrix synthesis and breakdown. Deviations in mechanical load, nutrient supply, cellular activity, matrix composition and metabolism may initiate a cascade ultimately leading to tissue dehydration, fibrosis, nerve and vessel ingrowth, disc height loss and disc herniation. Spinal instability, inflammation and neural sensitisation are sources of back pain, a worldwide leading burden that is challenging to cure. In this review, advances in cell and molecular therapy, including mobilisation and activation of endogenous progenitor cells, progenitor cell homing, and targeted delivery of cells, genes, or bioactive factors are discussed. 

Advancing the cellular and molecular therapy for intervertebral disc disease.

Intervertebral disc (IVD) disorders, especially degenerative disc disease, reduce the quality of life, and are short of effective therapy. A new direction for treatment of chronic tissue and organ diseases is to promote regeneration by harnessing endogenous repair mechanisms. In this review, we discuss the potential of endogenous repair in the IVD, the recent findings on endogenous IVD progenitor cells, and stem cell niches involved in IVD endogenous repair. We also highlight the factors which may restrict IVD self-healing. Ultimately, advanced therapeutic attempts to boost endogenous repair in the IVD are discussed, including bioactive factor delivery, gene therapy, activation of endogenous IVD progenitor cells and chemokine mediated stem cell homing. 

Potential and limitations of intervertebral disc endogenous repair.

Nucleus pulposus (NP) replacement offers a minimally invasive alternative to spinal fusion or total disc replacement for the treatment of intervertebral disc (IVD) degeneration. This study aimed to develop a cytocompatible NP replacement material, which is feasible for non-invasive delivery and tunable design, and allows immediate mechanical restoration of the IVD. A bi-phasic polyurethane scaffold was fabricated consisting of a core material with rapid swelling property and a flexible electrospun envelope. The scaffold was assessed in a bovine whole IVD organ culture model under dynamic load for 14 days. Nucleotomy was achieved by incision through the endplate without damaging the annulus fibrosus. After implantation of the scaffold and in situ swelling, the dynamic compressive stiffness and disc height were restored immediately. The scaffold also showed favorable cytocompatibility for native disc cells. Implantation of the scaffold in a partially nucleotomized IVD down-regulated catabolic gene expression, increased proteoglycan and type II collagen intensity and decreased type I collagen intensity in remaining NP tissue, indicating potential to retard degeneration and preserve the IVD cell phenotype. The scaffold can be delivered in a minimally invasive manner, and the geometry of the scaffold post-hydration is tunable by adjusting the core material, which allows individualized design. 

Polyurethane scaffold with in situ swelling capacity for nucleus pulposus replacement.

Surgical procedures such as microfracture or autologous chondrocyte implantation have been used to treat articular cartilage lesions; however, repair often fails in terms of matrix organization and mechanical behaviour. Advanced biomaterials and tissue engineered constructs have been developed to improve cartilage repair; nevertheless, their clinical translation has been hampered by the lack of reliable in vitro models suitable for pre-clinical screening of new implants and compounds. In this study, an osteochondral defect model in a bioreactor that mimics the multi-axial motion of an articulating joint, was developed. Osteochondral explants were obtained from bovine stifle joints, and cartilage defects of 4 mm diameter were created. The explants were used as an interface against a ceramic ball applying dynamic compressive and shear loading. Osteochondral defects were filled with chondrocytes-seeded fibrin-polyurethane constructs and subjected to mechanical stimulation. Cartilage viability, proteoglycan accumulation and gene expression of seeded chondrocytes were compared to free swelling controls. Cells within both cartilage and bone remained viable throughout the 10-day culture period. Loading did not wear the cartilage, as indicated by histological evaluation and glycosaminoglycan release. The gene expression of seeded chondrocytes indicated a chondrogenic response to the mechanical stimulation. Proteoglycan 4 and cartilage oligomeric matrix protein were markedly increased, while mRNA ratios of collagen type II to type I and aggrecan to versican were also enhanced. This mechanically stimulated osteochondral defect culture model provides a viable microenvironment and will be a useful pre-clinical tool to screen new biomaterials and biological regenerative therapies under relevant complex mechanical stimuli. STATEMENT OF SIGNIFICANCE: Articular cartilage lesions have a poor healing capacity and reflect one of the most challenging problems in orthopedic clinical practice. The aim of current research is to develop a testing system to assess biomaterials for implants, that can permanently replace damaged cartilage with the original hyaline structure and can withstand the mechanical forces long term. Here, we present an osteochondral ex vivo culture model within a cartilage bioreactor, which mimics the complex motion of an articulating joint in vivo. The implementation of mechanical forces is essential for pre-clinical testing of novel technologies in the field of cartilage repair, biomaterial engineering and regenerative medicine. Our model provides a unique opportunity to investigate healing of articular cartilage defects in a physiological joint-like environment.

Mechanically stimulated osteochondral organ culture for evaluation of biomaterials in cartilage repair studies.

This study aimed to characterize the mesenchymal stem cell (MSC) subpopulation migrating towards a degenerated intervertebral disc (IVD) and to assess its regenerative potential.

CD146/MCAM distinguishes stem cell subpopulations with distinct migration and regenerative potential in degenerative intervertebral discs.

We investigated the effects of a fibrin-hyaluronic acid hydrogel (FBG-HA) and fibroblast growth factor 18 (FGF-18) for nucleus pulposus (NP) regeneration. Healthy bovine ( = 4) and human degenerated NP cells ( = 4) were cultured for 14 days in FBG-HA hydrogel with FGF-18 (∆51-mutant or wild-type) in the culture medium. Gene expression, DNA content, and glycosaminoglycan (GAG) synthesis were evaluated on day 7 and 14. Additionally, histology was performed. Human NP cells cultured in FBG-HA hydrogel showed an increase in collagen type II (COL2) and carbonic anhydrase XII (CA12) gene expression after 14 or 7 days of culture, respectively. GAG release into the conditioned medium increased over 14 days. Healthy bovine NP cells showed increased gene expression of ACAN from day 7 to day 14. Wild type FGF-18 up-regulated CA12 gene expression of human NP cells. Histology revealed an increase of proteoglycan deposition upon FGF-18 stimulation in bovine but not in human NP cells. The FBG-HA hydrogel had a positive modulatory effect on human degenerated NP cells. Under the tested conditions, no significant effect of FGF-18 was observed on cell proliferation or GAG synthesis in human NP cells.

Fibrin-Hyaluronic Acid Hydrogel (RegenoGel) with Fibroblast Growth Factor-18 for In Vitro 3D Culture of Human and Bovine Nucleus Pulposus Cells.

Biomaterials play a pivotal role in cell-free cartilage repair approaches, where cells must migrate through the scaffold, fill the defect, and then proliferate and differentiate facilitating tissue remodeling. Here we used multiple assays to test the influence of chemokines and growth factors on cell migration and cartilage repair in two different hyaluronan (HA)-based hydrogels. We first investigated bone marrow Mesenchymal Stromal Cells (BMSC) migration in vitro, in response to different concentrations of platelet-derived growth factor-BB (PDGF-BB), chemokine ligand 5 (CCL5/RANTES) and stromal cell-derived factor 1 (SDF-1), using a 3D spheroid-based assay. PDGF-BB was selected as most favourable chemotactic agent, and MSC migration was assessed in the context of physical impediment to cell recruitment by testing Fibrin-HA and HA-Tyramine hydrogels of different cross-linking densities. Supplementation of PDGF-BB stimulated progressive migration of MSC through the gels over time. We then investigated in situ cell migration into the hydrogels with and without PDGF-BB, using a cartilage-bone explant model implanted subcutaneously in athymic mice. In vivo studies show that when placed into an osteochondral defect, both hydrogels supported endogenous cell infiltration and provided an amenable microenvironment for cartilage production. These processes were best supported in Fibrin-HA hydrogel in the absence of PDGF-BB. This study used an advanced preclinical testing platform to select an appropriate microenvironment provided by implanted hydrogels, demonstrating that HA-based hydrogels can promote the initial and critical step of endogenous cell recruitment and circumvent some of the clinical challenges in cartilage tissue repair. STATEMENT OF SIGNIFICANCE: The challenge of articular cartilage repair arises from its complex structure and architecture, which confers the unique mechanical behavior of the extracellular matrix. The aim of our research is to identify biomaterials for implants that can support migration of endogenous stem and progenitor cell populations from cartilage and bone tissue, in order to permanently replace damaged cartilage with the original hyaline structure. Here, we present an in vitro 3D spheroid-based migration assay and an osteochondral defect model, which provide the opportunity to assess biomaterials and biomolecules, and to get stronger experimental evidence of the not well-characterized dynamic process of endogenous cells colonization in an osteochondral defect. Furthermore, the delicate step of early cell migration into biomaterials towards functional tissue engineering is reproduced. These tests can be used for pre-clinical testing of newly developed material designs in the field of scaffold engineering.

Evaluation of biomimetic hyaluronic-based hydrogels with enhanced endogenous cell recruitment and cartilage matrix formation.

Injury of articular cartilage leads to an imbalance in tissue homeostasis, and due to the poor self-healing capacity of cartilage the affected tissue often exhibits osteoarthritic changes. In recent years, injectable and highly tunable composite hydrogels for cartilage tissue engineering and drug delivery have been introduced as a desirable alternative to invasive treatments. In this study, we aimed to formulate injectable hydrogels for drug delivery and cartilage tissue engineering by combining different concentrations of hyaluronic acid-tyramine (HA-Tyr) with regenerated silk-fibroin (SF) solutions. Upon enzymatic crosslinking, the gelation and mechanical properties were characterized over time. To evaluate the effect of the hydrogel compositions and properties on extracellular matrix (ECM) deposition, bovine chondrocytes were embedded in enzymatically crosslinked HA-Tyr/SF composites (in further work abbreviated as HA/SF) or HA-Tyr hydrogels. We demonstrated that all hydrogel formulations were cytocompatible and could promote the expression of cartilage matrix proteins allowing chondrocytes to produce ECM, while the most prominent chondrogenic effects were observed in hydrogels with HA20/SF80 polymeric ratios. Unconfined mechanical testing showed that the compressive modulus for HA20/SF80 chondrocyte-laden constructs was increased almost 10-fold over 28 days of culture in chondrogenic medium which confirmed the superior production of ECM in this hydrogel compared to other hydrogels in this study. Furthermore, in hydrogels loaded with anabolic and anti-inflammatory drugs, HA20/SF80 hydrogel showed the longest and the most sustained release profile over time which is desirable for the long treatment duration typically necessary for osteoarthritic joints. In conclusion, HA20/SF80 hydrogel was successfully established as a suitable injectable biomaterial for cartilage tissue engineering and drug delivery applications.

Optimization of hyaluronic acid-tyramine/silk-fibroin composite hydrogels for cartilage tissue engineering and delivery of anti-inflammatory and anabolic drugs.

Article	Year
Chondrocyte gene expression under applied surface motion. Biorheology\| PubMed ID: 16912399	2006
Effects of simple and complex motion patterns on gene expression of chondrocytes seeded in 3D scaffolds. Tissue engineering\| PubMed ID: 17518631	2006
[Chondrocytes - one cell type, different subpopulations : characteristics and behavior of different types of chondrocytes and implications for tissue engineering applications]. Der Orthopade\| PubMed ID: 19802604	2009
Sliding motion modulates stiffness and friction coefficient at the surface of tissue engineered cartilage. Osteoarthritis and cartilage\| PubMed ID: 22285735	2012
The effect of hyaluronan-based delivery of stromal cell-derived factor-1 on the recruitment of MSCs in degenerating intervertebral discs. Biomaterials\| PubMed ID: 24969636	2014
Advancing the cellular and molecular therapy for intervertebral disc disease. Advanced drug delivery reviews\| PubMed ID: 24993611	2015
Potential and limitations of intervertebral disc endogenous repair. Current stem cell research & therapy\| PubMed ID: 25741710	2015
Polyurethane scaffold with in situ swelling capacity for nucleus pulposus replacement. Biomaterials\| PubMed ID: 26828684	2016
Mechanically stimulated osteochondral organ culture for evaluation of biomaterials in cartilage repair studies. Acta biomaterialia\| PubMed ID: 30273741	2018
CD146/MCAM distinguishes stem cell subpopulations with distinct migration and regenerative potential in degenerative intervertebral discs. Osteoarthritis and cartilage\| PubMed ID: 31002939	2019
Fibrin-Hyaluronic Acid Hydrogel (RegenoGel) with Fibroblast Growth Factor-18 for In Vitro 3D Culture of Human and Bovine Nucleus Pulposus Cells. International journal of molecular sciences\| PubMed ID: 31614494	2019
Evaluation of biomimetic hyaluronic-based hydrogels with enhanced endogenous cell recruitment and cartilage matrix formation. Acta biomaterialia\| PubMed ID: 31726249	2020
Optimization of hyaluronic acid-tyramine/silk-fibroin composite hydrogels for cartilage tissue engineering and delivery of anti-inflammatory and anabolic drugs. Materials science & engineering. C, Materials for biological applications\| PubMed ID: 33545860	2021

Sibylle Grad

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