Marianna Peroglio

Diversity of intervertebral disc cells: phenotype and function

The intervertebral disc (IVD) is a moderately moving joint that is located between the bony vertebrae and provides flexibility and load transmission throughout the spinal column. The disc is composed of different but interrelated tissues, including the central highly hydrated nucleus pulposus (NP), the surrounding elastic and fibrous annulus fibrosus (AF), and the cartilaginous endplate (CEP), which provides the connection to the vertebral bodies. Each of these tissues has a different function and consists of a specific matrix structure that is maintained by a cell population with distinct phenotype. Although the healthy IVD is able to balance the slow matrix turnover of synthesis and degradation, this balance is often disturbed, leading to degenerative disorders. Successful therapeutic management of IVD degeneration requires a profound understanding of the cellular and molecular characteristics of the functional IVD. Hence, the phenotype of IVD cells has been of significant interest from multiple perspectives, including development, growth, remodelling, degeneration and repair. One major challenge that complicates our understanding of the disc cells is that both the cellular phenotype and the extracellular matrix strongly depend on disc maturity and health and as a consequence are continuously evolving. This review delineates the diversity of the cell types found in the intervertebral disc, with emphasis on human, but with reference to other species. The cells of the NP appear rounded and express a proteoglycan‐rich matrix, whereas the more elongated AF cells are embedded in a collagen fibre matrix and the CEPs represent a layer of cartilage. Even though all disc cells have often been referred to as ‘intervertebral disc chondrocytes’, distinct phenotypical differences in comparison with articular chondrocytes exist and have been reported recently. The availability of more specific markers has also improved our understanding of progenitor cell differentiation towards an IVD cell phenotype. Ultimately, new cell‐ and tissue‐engineering approaches to regenerative therapies will only be successful if the specific characteristics of the individual tissues and their context in the function of the whole organ, are taken into consideration.

Mechanical properties and cytocompatibility of poly(ε-caprolactone)-infiltrated biphasic calcium phosphate scaffolds with bimodal pore distribution

Biphasic calcium phosphate scaffolds have attracted interest because they have good osteoconductivity and a resorption rate close to that of new bone ingrowth, but their brittleness limits their potential applications. In this study, we show how the infiltration of biphasic calcium phosphate scaffolds with poly(ε-caprolactone) improves their mechanical properties. It was found that the polymer effectively contributes to energy to failure enhancement in bending, compressive and tensile tests. The main toughening mechanism in these composites is crack bridging by polymer fibrils. The presence of fibrils at two different size scales--as found in scaffolds with a bimodal pore distribution--results in a more effective toughening effect as compared to scaffolds with a monomodal pore size distribution, especially in the early stage of mechanical deformation. An optimized infiltration process allowed the preservation of micropore interconnection after infiltration, which is beneficial for cells adhesion. In addition, it is shown that biphasic calcium phosphates infiltrated with poly(ε-caprolactone) are cytocompatible with human bone marrow stromal cells, which makes them good candidates for bone substitution.

Thermoreversible hyaluronan-based hydrogel supports in vitro and ex vivo disc-like differentiation of human mesenchymal stem cells

BACKGROUND CONTEXT The fate of human mesenchymal stem cells (hMSCs) supplied to the degenerating intervertebral disc (IVD) is still not fully understood and can be negatively affected by low oxygen, pH, and glucose concentration of the IVD environment. The hMSC survival and yield upon injection of compromised IVD could be improved by the use of an appropriate carrier and/or by predifferentiation of hMSCs before injection. PURPOSE To optimize hMSC culture conditions in thermoreversible hyaluronan-based hydrogel, hyaluronan-poly(N-isopropylacrylamide) (HA-pNIPAM), to achieve differentiation toward the disc phenotype in vitro, and evaluate whether preconditioning contributes to a better hMSC response ex vivo. STUDY DESIGN In vitro and ex vivo whole-organ culture of hMSCs. METHODS In vitro cultures of hMSCs were conducted in HA-pNIPAM and alginate for 1 week under hypoxia in chondropermissive medium alone and with the supplementation of transforming growth factor β1 or growth and differentiation factor 5 (GDF-5). Ex vivo, hMSCs were either suspended in HA-pNIPAM and directly supplied to the IVDs or predifferentiated with GDF-5 for 1 week in HA-pNIPAM and then supplied to the IVDs. Cell viability was evaluated by Live-Dead assay, and DNA, glycosaminoglycan (GAG), and gene expression profiles were used to assess hMSC differentiation toward the disc phenotype. RESULTS The HA-pNIPAM induced hMSC differentiation toward the disc phenotype more effectively than alginate: in vitro, higher GAG/DNA ratio and higher collagen type II, SOX9, cytokeratin-19, cluster of differentiation 24, and forkhead box protein F1 expressions were found for hMSCs cultured in HA-pNIPAM compared with those cultured in alginate, regardless of the addition of growth factors. Ex vivo, direct combination of HA-pNIPAM with the disc environment induced a stronger disc-like differentiation of hMSCs than predifferentiation of hMSCs followed by their delivery to the discs. CONCLUSIONS Hyaluronan-based thermoreversible hydrogel supports hMSC differentiation toward the disc phenotype without the need for growth factor supplementation in vitro and ex vivo. Further in vivo studies are required to confirm the suitability of this hydrogel as an effective stem cell carrier for the treatment of IVD degeneration.

Homing of Mesenchymal Stem Cells in Induced Degenerative Intervertebral Discs in a Whole Organ Culture System

Study Design. Homing of human bone marrow–derived mesenchymal stem cells (BMSCs) was studied using ex vivo cultured bovine caudal intervertebral discs (IVDs). Objective. To investigate in a whole organ culture whether metabolic and mechanical challenges can induce BMSC recruitment into the IVD. Summary of Background Data. Cells from injured tissues release cytokines and mediators that enable the recruitment of progenitor cells. BMSCs have the ability to survive within the IVD. Methods. Bovine IVDs with or without endplates were cultured for 1 week under simulated physiological or degenerative conditions; disc cells were analyzed for cell viability and gene expression, whereas media was analyzed for nitric oxide production and chemotaxis. Homing of BMSCs was investigated by supplying PKH-labeled human BMSCs onto cultured IVDs (1 × 106 cells/disc on d 8, 10, and 12 of culture); on day 14, the number of homed BMSCs was microscopically assessed. Moreover, a comparative study was performed between transduced BMSCs (transduced with an adenovirus encoding for insulin-like growth factor 1 [IGF-1]) and nontransduced BMSCs. Disc proteoglycan synthesis rate was quantified via 35S incorporation. The secretion of IGF-1 was evaluated by enzyme-linked immunosorbent assay on both simulated physiological and degenerative discs. Results. Discs cultured under degenerative conditions showed reduced cell viability, upregulation of matrix degrading enzymes, and increased nitric oxide production compared with simulated physiological discs. Greater homing occurred under degenerative compared with physiological conditions with or without endplate. Media of degenerative discs demonstrated a chemoattractive activity toward BMSCs. Finally, discs homed with IGF-1–transduced BMSCs showed increased IGF-1 secretion and significantly higher proteoglycan synthesis rate than discs supplied with nontransduced BMSCs. Conclusion. We have demonstrated for the first time that degenerative conditions induce the release of factors promoting BMSC recruitment in an ex vivo organ culture. Moreover, IGF-1 transduction of BMSCs strongly increases the rate of proteoglycan synthesis within degenerative discs. This finding offers a new delivery system for BMSCs and treatment strategy for IVD regeneration.

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

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 transpedicular approach as an alternative route for intervertebral disc regeneration.

Study Design. Descriptive anatomical study on ovine and human cadaveric lumbar spinal segments. Objective. To describe the alternative transpedicular approach to deliver therapeutic agents into intervertebral disc (IVD). Summary of Background Data. The present delivery approach of therapeutic agents (growth factors/cells/hydrogels) within the IVD is through injection, via the annulus fibrosus (AF). However, it has recently been demonstrated that small needle puncture of the AF leads to further degeneration and disc herniation. In addition, the injected material has a high chance to be extruded through the AF injury. Methods. Lumbar ovine and human spinal segments were used. Under fluoroscopy, a 2-mm Kirschner wire was introduced in the caudal vertebra through the pedicle and the inferior endplate to the nucleus pulposus. Gross anatomy analysis and high-resolution peripheral quantitative computed tomography (HR-pQCT) were performed to assess the right position of the wire in pedicles. Discography and nucleotomy were performed using a 14G cannula insertion or a 2-mm arthroscopic shaver blade, respectively. Nucleoplasty was also performed with agarose gel/contrast agent and imaged with HR-pQCT. Results. Gross anatomy, fluoroscopy, and HR-pQCT images showed that the nucleus pulposus could be approached through the endplate via the pedicle without affecting the spinal canal and the neural foramina. The contrast agent was delivered into the IVD and nucleus pulposus was removed from the disc and filled with agarose gel. Conclusion. This study describes how a transpedicular approach can be used as an alternative route to deliver therapeutic agents to the disc without disruption of the AF showing the potential use of this technique in preclinical research and highlighting its clinical relevance for IVD regeneration.

Biomimetic fibrin-hyaluronan hydrogels for nucleus pulposus regeneration.

AIM To develop a biomimetic polymeric injectable hydrogel that can support nucleus pulposus (NP) regeneration. MATERIALS & METHODS Natural polymer-based hydrogels were synthesized using fibrinogen (FBG) and hyaluronic acid (HA), conjugated by a novel two-step procedure. Bovine NP cells were cultured in FBG-HA conjugate-based 3D beads in vitro and in a nucleotomized organ culture model. RESULTS FBG-HA conjugate-based hydrogels prepared with 235 KDa HA at a FBG/HA w/w ratio of 17:1 showed superior gel stability and mechanical properties and markedly increased glycosaminoglycan synthesis compared with a FBG/HA mixture-based hydrogels or fibrin gels. Gene-expression levels of NP markers were maintained in vitro. In organ culture, NP cells seeded in FBG-HA conjugate-based hydrogels showed better integration with native NP tissue compared with fibrin gels. Moreover, FBG-HA conjugate-based hydrogels restored compressive stiffness and disc height after nucleotomy under dynamic load. CONCLUSION Specific FBG-HA conjugate-based hydrogels may be suitable as injectable materials for minimally invasive, biological NP regeneration.

The osteogenic differentiation of human osteoprogenitor cells on Anodic-Plasma-Chemical treated Ti6Al7Nb.

Biological integration of an implant to surrounding bone is an important event for its clinical success and is driven by numerous factors, including the attraction of bone forming cells. The implants surface properties influence the initial cell response at the cell/material interface, ultimately affecting the rate and quality of new tissue formation and the stability of the implant. As a consequence, various surface treatments have been developed to increase the clinical performance of titanium-based implants. Among them, the Anodic Plasma-Chemical (APC) technique allows for the combined chemical and morphological modification of titanium surfaces in a single process step. In the present study, we compared the potential of APC surface treatment of high-strength titanium alloys with vacuum plasma spray treatment and yellow gold anodization in supporting osteogenic differentiation of two different osteoprogenitor cell types. Both human fetal osteoblast cell line (hFOB1.19) and human mesenchymal stromal cells showed extensive cell spreading, faster cell growth and differentiation on APC surfaces compared to vacuum plasma spray treated and yellow gold anodized surfaces. Our findings showed that APC titanium-based surfaces provided an effective substrate for osteoprogenitor cells adhesion, proliferation and differentiation.

Self-Healing Dynamic Hydrogel as Injectable Shock-Absorbing Artificial Nucleus Pulposus

The intervertebral discs (IVDs) provide unique flexibility to the spine and exceptional shock absorbing properties under impact. The inner core of the IVD, the nucleus pulposus (NP) is responsible for this adaptive behavior. Herein, we evaluate an injectable, self-healing dynamic hydrogel (DH) based on gold(I)-thiolate/disulfide (Au-S/SS) exchange as NP replacement in a spine motion segment model. For the first time, we report the application of dynamic covalent hydrogels inside biological tissues. The dynamic exchange between Au-S species and disulfide bonds (SS) resulted in self-healing ability and frequency-dependent stiffness of the hydrogel, which was also confirmed in spine motion segments. Injection of preformed DH into nucleotomized IVDs restored the full biomechanical properties of intact IVDs, including the stiffening effect observed at increasing frequencies, which cannot be achieved with conventional covalent hydrogel. DH has the potential to counteract IVD degeneration associated with high frequency vibrations. Self-healing properties, confirmed by rheology studies and macroscopic observation after injection, were required to inject preformed DH, which recovered its mechanical integrity and microstructure to act as an artificial NP. On the other hand, covalent hydrogel did not show any restoration of NP properties as this conventional material suffered irreversible damages after injection, which demonstrates that the dynamic properties are crucial for this application. The persistence of DH in the IVD space following cyclic high-frequency loading, confirmed by tomography after mechanical testing, suggests that this material would have long life span as an injectable NP replacement material.

Multivalent dendrimers presenting spatially controlled clusters of binding epitopes in thermoresponsive hyaluronan hydrogels

The controlled presentation of biofunctionality is of key importance for hydrogel applications in cell-based regenerative medicine. Here, a versatile approach was demonstrated to present clustered binding epitopes in an injectable, thermoresponsive hydrogel. Well-defined multivalent dendrimers bearing four integrin binding sequences and an azido moiety were covalently grafted to propargylamine-derived hyaluronic acid (Hyal-pa) using copper-catalyzed alkyne-azide cycloaddition (CuAAC), and then combined with pN-modified hyaluronan (Hyal-pN). The dendrimers were prepared by synthesizing a bifunctional diethylenetriamine pentaacetic acid core with azido and NHBoc oligo(ethylene glycol) aminoethyl branches, then further conjugated with solid-phase synthesized RGDS and DGRS peptides. Azido terminated pN was synthesized by reversible addition-fragmentation chain transfer polymerization and reacted to Hyal-pa via CuAAC. Nuclear magnetic resonance (NMR), high performance liquid chromatography, size exclusion chromatography and mass spectroscopy proved that the dendrimers had well-defined size and were disubstituted. NMR and atomic absorption analysis confirmed the hyaluronan was affixed with dendrimers or pN. Rheological measurements demonstrated that dendrimers do not influence the elastic or viscous moduli of thermoresponsive hyaluronan compositions at a relevant biological concentration. Finally, human mesenchymal stromal cells were encapsulated in the biomaterial and cultured for 21days, demonstrating the faculty of this dendrimer-modified hydrogel as a molecular toolbox for tailoring the biofunctionality of thermoresponsive hyaluronan carriers for biomedical applications.