Frances C. Bach

The paracrine feedback loop between vitamin D₃ (1,25(OH)₂D₃) and PTHrP in prehypertrophic chondrocytes.

The endocrine feedback loop between vitamin D3 (1,25(OH)2D3) and parathyroid hormone (PTH) plays a central role in skeletal development. PTH‐related protein (PTHrP) shares homology and its receptor (PTHR1) with PTH. The aim of this study was to investigate whether there is a functional paracrine feedback loop between 1,25(OH)2D3 and PTHrP in the growth plate, in parallel with the endocrine feedback loop between 1,25(OH)2D3 and PTH. This was investigated in ATDC5 cells treated with 10−8 M 1,25(OH)2D3 or PTHrP, Col2‐pd2EGFP transgenic mice, and primary Col2‐pd2EGFP growth plate chondrocytes isolated by FACS, using RT‐qPCR, Western blot, PTHrP ELISA, chromatin immunoprecipitation (ChIP) assay, silencing of the 1,25(OH)2D3 receptor (VDR), immunofluorescent staining, immunohistochemistry, and histomorphometric analysis of the growth plate. The ChIP assay confirmed functional binding of the VDR to the PTHrP promoter, but not to the PTHR1 promoter. Treatment with 1,25(OH)2D3 decreased PTHrP protein production, an effect which was prevented by silencing of the VDR. Treatment with PTHrP significantly induced VDR production, but did not affect 1α‐ and 24‐hydroxylase expression. Hypertrophic differentiation was inhibited by PTHrP and 1,25(OH)2D3 treatment. Taken together, these findings indicate that there is a functional paracrine feedback loop between 1,25(OH)2D3 and PTHrP in the growth plate. 1,25(OH)2D3 decreases PTHrP production, while PTHrP increases chondrocyte sensitivity to 1,25(OH)2D3 by increasing VDR production. In light of the role of 1,25(OH)2D3 and PTHrP in modulating chondrocyte differentiation, 1,25(OH)2D3 in addition to PTHrP could potentially be used to prevent undesirable hypertrophic chondrocyte differentiation during cartilage repair or regeneration. J. Cell. Physiol. 229: 1999–2014, 2014.

Link-N:the missing link towards intervertebral disc repair is species-specific

Introduction Degeneration of the intervertebral disc (IVD) is a frequent cause for back pain in humans and dogs. Link-N stabilizes proteoglycan aggregates in cartilaginous tissues and exerts growth factor-like effects. The human variant of Link-N facilitates IVD regeneration in several species in vitro by inducing Smad1 signaling, but it is not clear whether this is species specific. Dogs with IVD disease could possibly benefit from Link-N treatment, but Link-N has not been tested on canine IVD cells. If Link-N appears to be effective in canines, this would facilitate translation of Link-N into the clinic using the dog as an in vivo large animal model for human IVD degeneration. Materials and methods This study’s objective was to determine the effect of the human and canine variant of Link-N and short (s) Link-N on canine chondrocyte-like cells (CLCs) and compare this to those on already studied species, i.e. human and bovine CLCs. Extracellular matrix (ECM) production was determined by measuring glycosaminoglycan (GAG) content and histological evaluation. Additionally, the micro-aggregates’ DNA content was measured. Phosphorylated (p) Smad1 and -2 levels were determined using ELISA. Results Human (s)Link-N induced GAG deposition in human and bovine CLCs, as expected. In contrast, canine (s)Link-N did not affect ECM production in human CLCs, while it mainly induced collagen type I and II deposition in bovine CLCs. In canine CLCs, both canine and human (s)Link-N induced negligible GAG deposition. Surprisingly, human and canine (s)Link-N did not induce Smad signaling in human and bovine CLCs. Human and canine (s)Link-N only mildly increased pSmad1 and Smad2 levels in canine CLCs. Conclusions Human and canine (s)Link-N exerted species-specific effects on CLCs from early degenerated IVDs. Both variants, however, lacked the potency as canine IVD regeneration agent. While these studies demonstrate the challenges of translational studies in large animal models, (s)Link-N still holds a regenerative potential for humans.

Biologic canine and human intervertebral disc repair by notochordal cell-derived matrix : From bench towards bedside

The socioeconomic burden of chronic back pain related to intervertebral disc (IVD) disease is high and current treatments are only symptomatic. Minimally invasive strategies that promote biological IVD repair should address this unmet need. Notochordal cells (NCs) are replaced by chondrocyte-like cells (CLCs) during IVD maturation and degeneration. The regenerative potential of NC-secreted substances on CLCs and mesenchymal stromal cells (MSCs) has already been demonstrated. However, identification of these substances remains elusive. Innovatively, this study exploits the regenerative NC potential by using healthy porcine NC-derived matrix (NCM) and employs the dog as a clinically relevant translational model. NCM increased the glycosaminoglycan and DNA content of human and canine CLC aggregates and facilitated chondrogenic differentiation of canine MSCs in vitro. Based on these results, NCM, MSCs and NCM+MSCs were injected in mildly (spontaneously) and moderately (induced) degenerated canine IVDs in vivo and, after six months of treatment, were analyzed. NCM injected in moderately (induced) degenerated canine IVDs exerted beneficial effects at the macroscopic and MRI level, induced collagen type II-rich extracellular matrix production, improved the disc height, and ameliorated local inflammation. MSCs exerted no (additive) effects. In conclusion, NCM induced in vivo regenerative effects on degenerated canine IVDs. NCM may, comparable to demineralized bone matrix in bone regeneration, serve as ‘instructive matrix’, by locally releasing growth factors and facilitating tissue repair. Therefore, intradiscal NCM injection could be a promising regenerative treatment for IVD disease, circumventing the cumbersome identification of bioactive NC-secreted substances.

Leaping the hurdles in developing regenerative treatments for the intervertebral disc from preclinical to clinical

Chronic back and neck pain is a prevalent disability, often caused by degeneration of the intervertebral disc. Because current treatments for this condition are less than satisfactory, a great deal of effort is being applied to develop new solutions, including regenerative strategies. However, the path from initial promising idea to clinical use is fraught with many hurdles to overcome. Many of the keys to success are not necessarily linked to science or innovation. Successful translation to clinic will also rely on planning and awareness of the hurdles. It will be essential to plan your entire path to clinic from the outset and to do this with a multidisciplinary team. Take advice early on regulatory aspects and focus on generating the proof required to satisfy regulatory approval. Scientific demonstration and societal benefits are important, but translation cannot occur without involving commercial parties, which are instrumental to support expensive clinical trials. This will only be possible when intellectual property can be protected sufficiently to support a business model. In this manner, commercial, societal, medical, and scientific partners can work together to ultimately improve patient health. Based on literature surveys and experiences of the co‐authors, this opinion paper presents this pathway, highlights the most prominent issues and hopefully will aid in your own translational endeavors.

Notochordal-cell derived extracellular vesicles exert regenerative effects on canine and human nucleus pulposus cells

During intervertebral disc ageing, chondrocyte-like cells (CLCs) replace notochordal cells (NCs). NCs have been shown to induce regenerative effects in CLCs. Since vesicles released by NCs may be responsible for these effects, we characterized NC-derived extracellular vesicles (EVs) and determined their effect on CLCs. EVs were purified from porcine NC-conditioned medium (NCCM) through size exclusion chromatography, ultracentrifugation or density gradient centrifugation. Additionally, the EVs were quantitatively analyzed by high-resolution flow cytometry. The effect of NCCM-derived EVs was studied on canine and human CLC micro-aggregates in vitro and compared with NCCM-derived proteins and unfractionated NCCM. Porcine NCCM contained a considerable amount of EVs. NCCM-derived EVs induced GAG deposition in canine CLCs to a comparable level as NCCM-derived proteins and unfractionated NCCM, and increased the DNA and glycosaminoglycan (GAG) content of human micro-aggregates, although to a lesser extent than unfractionated NCCM. The biological EV effects were not considerably influenced by ultracentrifugation compared with size exclusion-based purification. Upon ultracentrifugation, interfering GAGs, but not collagens, were lost. Nonetheless, collagen type I or II supplemented to CLCs in a concentration as present in NCCM induced no anabolic effects. Porcine NCCM-derived EVs exerted anabolic effects comparable to NCCM-derived proteins, while unfractionated NCCM was more potent in human CLCs. GAGs and collagens appeared not to mediate the regenerative EV effects. Thus, NC-derived EVs have regenerative potential, and their effects may be influenced by the proteins present in NCCM. The optimal combination of NC-secreted factors needs to be determined to fully exploit the regenerative potential of NC-based technology.

Intradiscal delivery of celecoxib-loaded microspheres restores intervertebral disc integrity in a preclinical canine model

ABSTRACT Low back pain, related to degeneration of the intervertebral disc (IVD), affects millions of people worldwide. Clinical studies using oral cyclooxygenase‐2 (COX‐2) inhibitors have shown beneficial effects, although side‐effects were reported. Therefore, intradiscal delivery of nonsteroidal anti‐inflammatory drugs can be an alternative treatment strategy to halt degeneration and address IVD‐related pain. In the present study, the controlled release and biologic potency of celecoxib, a selective COX‐2 inhibitor, from polyesteramide microspheres was investigated in vitro. In addition, safety and efficacy of injection of celecoxib‐loaded microspheres were evaluated in vivo in a canine IVD degeneration model. In vitro, a sustained release of celecoxib was noted for over 28 days resulting in sustained inhibition of inflammation, as indicated by decreased prostaglandin E2 (PGE2) production, and anti‐catabolic effects in nucleus pulposus (NP) cells from degenerated IVDs on qPCR. In vivo, there was no evidence of adverse effects on computed tomography and magnetic resonance imaging or macroscopic evaluation of IVDs. Local and sustained delivery of celecoxib prevented progression of IVD degeneration corroborated by MRI, histology, and measurement of NP proteoglycan content. Furthermore, it seemed to harness inflammation as indicated by decreased PGE2 tissue levels and decreased neuronal growth factor immunopositivity, providing indirect evidence that local delivery of a COX‐2 inhibitor could also address pain related to IVD degeneration. In conclusion, intradiscal controlled release of celecoxib from polyesteramide microspheres prevented progression of IVD degeneration both in vitro and in vivo. Follow‐up studies are warranted to determine the clinical efficacy of celecoxib‐loaded PEAMs in chronic back pain. Graphical abstract Figure. No caption available.

Successful fishing for nucleus pulposus progenitor cells of the intervertebral disc across species

Recently, Tie2/TEK receptor tyrosine kinase (Tie2 or syn. angiopoietin‐1 receptor) positive nucleus pulposus progenitor cells were detected in human, cattle, and mouse. These cells show remarkable multilineage differentiation capacity and direct correlation with intervertebral disc (IVD) degeneration and are therefore an interesting target for regenerative strategies. Nevertheless, there remains controversy over the presence and function of these Tie2+ nucleus pulposus cells (NPCs), in part due to the difficulty of identification and isolation.

The Role of Caveolin-1 in Intervertebral Disc Degeneration and Regeneration

Introduction During intervertebral disc (IVD) degeneration, the main cell type in the nucleus pulposus (NP) shifts from notochordal cells (NCs) to chondrocyte-like cells (CLCs). Microarray analysis revealed that caveolin-1 expression was correlated with IVD degeneration. The aim of this study was to determine the role of caveolin-1 in NC and CLC physiology to assess its potential role in IVD regeneration. Material and Methods Protein expression (caveolin-1, apoptosis, progenitor cell markers, extracellular matrix, TGF-β-signaling pathway) was determined in IVDs of wild type (WT) and caveolin-1 knockout (KO) mice and canine IVDs of different degeneration grades (immunofluorescence, immunohistochemistry, TUNEL assay). Micro-aggregate cultures of CLCs from canine and human degenerated IVDs (Thompson grade III) were treated with chondrogenic medium (incl. TGF-β1) alone or in combination with (a) caveolin-1 scaffolding domain peptide (CSD) and/or (b) siRNA against caveolin-1. DNA, glycosaminoglycan (GAG) content, collagen type I and II immunohistochemistry and gene expression profiling (RT-qPCR) for extracellular matrix production/degradation-, cell proliferation- and apoptosis markers was performed. Results The NP of WT mice was rich in viable NCs, whereas the NP of caveolin-1 KO mice contained more collagen type II-rich matrix and less cells together with an increased progenitor cell surface marker (Tie2+/GD2+) expression and a higher apoptotic activity. Caveolin-1 expression increased in the later stages of canine IVD degeneration, together with a significantly increased apoptotic activity. Caveolin-1 knockdown significantly decreased GAG deposition in the CLC aggregates (6–14%), whereas CSD treatment significantly rescued and increased GAG deposition (11–16%). Conclusion Caveolin-1 plays a crucial role in preservation of NCs, underscored by the NP phenotype of caveolin-1 KO mice. Caveolin-1 may be related with cell senescence given its increased expression in degenerated IVDs. However, caveolin-1 knockdown decreased extracellular matrix production, while CSD supplementation rescued this effect. The latter implies that CSD may be a useful disease modifying agent since it is known to influence degeneration-related signaling pathways (incl. TGF-β signaling). Altogether, this indicates that the increased caveolin-1 expression during IVD degeneration may also be a repair mechanism rather than being merely a senescence marker. Acknowledgment This work was supported by AOSpine Research Network grants (SRN2011_11, AOSPINE 106540) and the Dutch Arthritis Foundation (LLP22).

The Effect of Bone Morphogenetic Protein-2 on Chondrocyte-like Cells Derived from Degenerated Human and Canine Intervertebral Discs

Introduction Both humans and dogs experience low back pain, which is related to intervertebral disc (IVD) degeneration. Biologic repair of the degenerated IVD is mainly based on growth factors that exert anabolic matrix effects, and mesenchymal stromal cells (MSCs) to replenish the cell population of the degenerated IVD. Thus far, the anabolic effects of different growth factors have not been compared. The aim of this study was to study the effect of the frequently used growth factors transforming growth factor β 1 (TGF-β1) and bone morphogenetic protein-2 (BMP-2) on canine and human chondrocyte-like cells (CLCs) from degenerated IVDs alone and in combination with MSCs. Material and Methods CLCs from degenerated human, canine chondrodystrophic (CD) and non-chondrodystrophic (NCD) IVDs (Thompson score III) were cultured in micro-aggregates in base culture medium (negative control), or supplemented with TGF-β1 (10 ng/mL) or BMP-2 (100 or 250 ng/mL) for 28 days. The additive effect of MSCs was studied in CD CLCs. Canine male CD CLCs were cultured in an albumin-based hydrogel (3*106 cells/mL) with or without the addition of female bone marrow-derived MSCs (BMSCs) (CLC:BMSC 1:1) in control or 250 ng/mL BMP-2-supplemented culture medium for 28 days. Read out parameters were extracellular matrix (ECM) production (RT-qPCR, glycosaminoglycan (GAG) production, Safranin O/Fast Green staining, immunohistochemistry), cell proliferation (DNA content, RT-qPCR) and apoptosis (RT-qPCR). Results TGF-β1 treatment increased GAG deposition in human and canine CLC micro-aggregates, but also induced collagen type I deposition and a fibrotic rim. The latter was not observed in BMP-2-treated micro-aggregates. 250 ng/mL BMP-2 was more potent than 100 ng/mL BMP-2 in increasing GAG deposition and DNA content in canine and human micro-aggregates. Similarly, in the hydrogel culture system, BMP-2 induced GAG and collagen type II deposition and a higher DNA content compared with untreated controls. DNA and GAG content of BMSC+CLC hydrogels was higher than hydrogels with CLCs alone in the absence of BMP-2. In the BMP-2-treated hydrogels, DNA content of BMSC+CLC was higher than hydrogels with CLCs alone; GAG deposition or release was comparable between BMSC+CLC and CLC alone in the presence of BMP-2. Conclusion In two different 3D culture systems, BMP-2 exerted comparable regenerative effects as TGF-β1 on human and canine CLCs in terms of GAG deposition and cell proliferation, but BMP-2 did not induce fibrotic (re)differentiation as observed with TGF-β1 treatment. Moreover, in the BMP-2-treated BMSC:CLC-containing hydrogels, where only half of the amount of CLCs were seeded compared with CLC alone, the DNA content was higher and an equal amount of GAGs was deposited, indicating that the BMSCs exerted an additional, regenerative effect in addition to BMP-2 treatment. PCR for SRY:GAPDH genes on DNA will indicate the ratio male(CLCs):female(BMSCs) present at the end of the study. This will indicate whether the BMSCs exerted trophic effects or chondrogenically differentiated. Acknowledgments This work was supported by an AOSpine Research Network grant (SRN2011_11) and the Dutch Arthritis Foundation (LLP22).