Mesenchymal stem cells (MSC) have potent immunomodulatory and regenerative effects via soluble factors. One approach to improve stem cell-based therapies is encapsulation of MSC in hydrogels based on natural proteins such as collagen and fibrin, which play critical roles in bone healing. In this work, we comparatively studied the influence of collagen and fibrin hydrogels of varying stiffness on the paracrine interactions established by MSC with macrophages and osteoblasts. Type I collagen and fibrin hydrogels in a similar stiffness range loaded with MSC from donants were prepared by modifying the protein concentration. Viability and morphology of MSC in hydrogels as well as cell migration rate from the matrices were determined. Paracrine actions of MSC in hydrogels were evaluated in co-cultures with human macrophages from healthy blood donors or with osteoblasts from bone explants of patients with osteonecrosis of the femoral head. Lower matrix stiffness resulted in higher MSC viability and migration. Cell migration rate from collagen hydrogels was higher than from fibrin matrices. The secretion of the immunomodulatory factors interleukin-6 (IL-6) and prostaglandin E. 2. (PGE. 2. ) by MSC in both collagen and fibrin hydrogels increased with increasing matrix stiffness. Tumor necrosis factor-α (TNF-α) secretion by macrophages cultured on collagen hydrogels was lower than on fibrin matrices. Interestingly, higher collagen matrix stiffness resulted in lower secreted TNF-α while the trend was opposite on fibrin hydrogels. In all cases, TNF-α levels were lower when macrophages were cultured on hydrogels containing MSC than on empty gels, an effect partially mediated by PGE. 2. Finally, mineralization capacity of osteoblasts co-cultured with MSC in hydrogels increased with increasing matrix stiffness, although this effect was more notably for collagen hydrogels. Paracrine interactions established by MSC in hydrogels with macrophages and osteoblasts are regulated by matrix composition and stiffness

Introduction and Objective. Mesenchymal stem cells (MSC) are attractive candidates for bone regeneration approaches. Benefits of MSC therapy are mainly attributed to paracrine effects via soluble factors, exerting both immunoregulatory and regenerative actions. Encapsulation of MSC in hydrogels prepared with extracellular matrix (ECM) proteins has been proposed as a strategy to enhance their survival and potentiate their function after implantation. Functional activity of MSC can be regulated by the physical and mechanical properties of their microenvironment. In this work, we investigated whether matrix stiffness can modulate the crosstalk between MSC encapsulated in collagen hydrogels with macrophages and osteoblasts. Materials and Method. Collagen hydrogels with a final collagen concentration of 1.5, 3 and 6 mg/mL loaded with human MSC were prepared. Viscoelastic properties of hydrogels were measured in a controlled stress rheometer. Cell distribution into the hydrogels was examined using confocal microscopy and the levels of the immunomodulatory factors interleukin-6 (IL-6) and prostaglandin E. 2. (PGE. 2. ) released by MSC were quantified by immunoassays. To determine the effect of matrix stiffness on the immunomodulatory potential of MSC, human macrophages obtained from healthy blood were cultured in media conditioned by MSC in hydrogels. The involvement of IL-6 and PGE. 2. in MSC-mediated immunomodulation was investigated employing neutralizing antibodies. Finally, the influence of soluble factors released by MSC in hydrogels on bone-forming cells was studied using osteoblasts obtained from trabecular bone explants from patients with osteonecrosis of the femoral head during total hip arthroplasty. Results. MSC loaded in hydrogels containing varying concentrations (1.5, 3 and 6 mg/mL) of collagen were viable. Rheology measurements determined that the hydrogel stiffness increased with increasing collagen concentration. Encapsulation of MSC into hydrogels barely affected their storage modulus values. MSC acquired a three-dimensional (3D) arrangement in all hydrogels and showed a more elongated shape in hydrogels with higher stiffness. The secretion of IL-6 and PGE. 2. by MSC in hydrogels increased with increasing matrix stiffness. Media conditioned by MSC encapsulated in stiffer hydrogels decreased TNF-α levels secreted by macrophages to a higher extent than media conditioned by MSC in softer hydrogels. This effect was partially mediated by PGE. 2. Finally, our preliminary results indicated that factors released by MSC in hydrogels regulated osteoblast-mediated mineralisation and this effect was dependent on hydrogel stiffness. Conclusions. Our data indicate that matrix stiffness of collagen hydrogels regulates the production of soluble factors by MSC and their paracrine actions on macrophages and osteoblasts

Vascular inflammation and activation of myofibroblasts are significant contributors to the progression of fibrosis, which can severely impair tissue function. In various tissues, including tendons, Transforming growth factor beta 1 (TGF-β1) has been identified as a critical driver of adhesion and scar formation. Nevertheless, the mechanisms that underlie fibrotic peritendinous adhesions are still not well comprehended, and human microphysiological systems to help identify effective therapies remain scarce. To address this issue, we developed a novel human Tendon-on-a-Chip (hToC), comprised of an endothelialized vascular compartment harboring circulating monocytes and separated by a 5 μm/100 nm dual-scale ultrathin porous membrane from a type I/III collagen hydrogel with primary tendon fibroblasts and tissue-resident macrophages, all under defined serum-free conditions. The hToC models the crosstalk of the various cells in the system leading to the induction of inflammatory and fibrotic pathways including the activation of mTOR signaling. Consistent with phenotypes observed in vivo in mouse models and clinical human samples, we observed myofibroblast differentiation and senescence, tissue contraction, excessive extracellular matrix deposition, and monocytes’ transmigration and macrophages’ secretion of inflammatory cytokines, which were dependent on the presence of the endothelial barrier. This model offers novel insights on the role of vasculature in the pathophysiology of adhesions, which were previously underappreciated. Moreover, in testing whether the hToC could be used to evaluate efficacy of therapeutics, we were able to capture donor-specific variability in the response to Rapamycin treatment, which reduced myofibroblast activation regardless. Thus, our findings demonstrate the value of the hToC as a human microphysiological system for investigating the pathophysiology of fibrotic conditions in the context of peritendinous injury and similar fibrotic conditions, providing an alternative to animal testing

INTRODUCTION. Intervertebral disc (IVD) degeneration is not completely understood because of the lack of relevant models. In vivo models are inappropriate because animals are quadrupeds. IVD is composed of the Nucleus Pulposus (NP) and the Annulus Fibrosus (AF), an elastic tissue that surrounds NP. AF consists of concentric lamellae made of collagen I and glycosaminoglycans with fibroblast-like cells located between layers. In this study, we aimed to develop a novel 3D in vitro model of Annulus Fibrosus to study its degeneration. For this purpose, we reproduced the microenvironment of AF cells using 3D printing. METHOD. An ink consisting of dense collagen (30 mg.mL. -1. ) and tyramine-functionalized hyaluronic acid (THA) at 7.5 mg.mL. -1. was first designed by modulating pH and [NaCl] in order to inhibit the formation of polyionic complexes between collagen and THA. Then, composite inks were printed in different gelling baths to form collagen hydrogels. Last, THA photocrosslinking using eosin and green light was performed to strengthen hydrogels. Selected 3D printed constructs were then cellularized with fibroblasts. RESULTS. The physicochemical study revealed that collagen/THA solutions (4:1 ratio) used at pH 5 with 200 mM NaCl were homogenous. In addition, collagen fibrils were observed in these solutions. The dense composite collagen/THA inks printed in a 2X PBS bath rapidly gelled and the photo-crosslinking increased the mechanical properties by 2 to reach 25 kPa (Young's modulus). Then, 3D printing parameters were optimized (85 kPa, extrusion, 4.5 mm/s speed and 80% fill-in percentage) to generate flat and anisotropic lamellae observed by polarized light microscopy. For the in vitro study, several anisotropic layers were printed and fibroblasts seeded between them. Cells adhered to layers, spread, proliferate and aligned along the axis of printed layers. CONCLUSION. Taken together, these results show it is possible to reproduce in vitro the main AF's biochemical and physical properties

First works focuses on the characterization (physical and biological) of this biomaterial. Current work had studied osteoinductive and osteoconductive capacity of these hydrogels. In vivoresults highlight a significant bone reconstruction two months after implantations on bone lesions in mice. Bone is a dynamic and vascularized tissue that has the ability of naturally healing upon damage. Nevertheless, in the case of critical size defects this potential is impaired. Present approaches mainly consider autografts and allografts, which presents several limitations. Bone Tissue Engineering (BTE) is based on the use of 3D matrices to guide both cellular growth, differentiation to promote bone regeneration. Hence, matrices can contain biological materials such as cells and growth factors. Our project aims to design a hydrogel for BTE, particularly for bone lesion filling. We previously showed that a porous 3D hydrogel, Glycosyl-Nucleoside-Fluorinated (GNF) is: 1) non-cytotoxic to clustered human Adipose Mesenchymal Stem Cells (hASCs), 2) bioinjectable and 3) biodegradable. Therefore, this novel class of hydrogels show promise for the development of therapeutic solutions for BTE [1]. The hypothesis of this research was that improving the capacity to promote the adhesion of cells by adding collagen gel matrices and bone morphogenic protein 2 (BMP-2) to improve the bone regenerative potential of this gel. Collagen is a protein matrix well known for its cytocompatibility [2]. BMP-2, have been shown ability to induce bone formation in combination with an adequate matrix [3]. Thereby, the overall aim of this work was to design, develop and validate a new composite hydrogel for BTE. GNF was prepared as previously described in detail[1], at a concentration of 3% (w/v). Type I-collagen gel was prepared from rat-tail tendons at a concentration of 4 g/L [2]. hASCs were isolated from human adipose tissue in our laboratory. To establish a suitable microenvironment for cell proliferation and differentiation cells were seeded in collagen and then GNF gel was added and the resulting mixture was blended, BMP-2 (InductOs ® Kit) is added to this preparation (5µm BMP-2/ml). Fluorometry was used to follow BMP2 release in vitro andin vivo(NOG mices;n=6), orthotopic calvariumbone critical defect (3.3 mm) has been selected to challenge the bone repair. Adding collagen hydrogel improve cell adhesion, survivals and proliferation rather than simple GNF hydrogel. This novel gel composite has the ability to sustain hASCs adhesion and differentiation towards the osteoblastic lineage (positive ALP cells). Fluorometry showed the ability of our hydrogel to prolong the residence of BMP-2 (in vitro and in vivo) compared to collagen hydrogel sponges. Implantation of hydrogel containing hASC and BMP-2 has shown encouraging results in bone reconstruction: 2 months after implantation of biomaterials a significant bone reconstruction can be observed using X-Ray imaging. Adding collagen to GNF allowed to obtain gels showing satisfactory cell-behaviour. In parallel, the presence of GNF hydrogel helps to improve mechanical properties of the biomaterial (hydrogel stability and controlled release of BMP-2). The first in vivostudies have shown encouraging bone regeneration capacity of these hydrogels. The implantation performed on a larger number of animals and quantitative microCT analysis will enable us to judge the effectiveness of this hydrogel as a new injectable biomaterial for BTE. This work was partially supported by NSERC-Canada, FRQ-NT-Quebec, FRQ-S- Quebec, and CFI-Canada. Mathieu Maisani was awarded of a NSERC CREATE Program in Regenerative Medicine (www.ncprm.ulaval.ca)

The extracellular matrix (ECM)-based biomaterials provide a platform to mimic the disc microenvironment in facilitating stem cell transplantation for tissue regeneration. However, little is known about in vitro preconditioning human umbilical cord Wharton Jelly-derived mesenchymal stem cells (MSCs) on 3D hyaluronic acid (HA)/type II collagen (COLII) hydrogel for nucleus pulposus (NP) phenotype and pain modulation. We developed a tuneable 3D HA/COLII by fabricating HA/COLII hydrogel at 2 mg/ml COLII and various weight ratios of HA:COLII, 1:9 and 4.5:9. The hydrogel was characterized for degradability, stability, and swelling capacity. The viability of hWJ-MSC encapsulated on hydrogel supplemented with TGF-β3 was assessed. The implantation of HA/COLII hydrogel was done in surgically induced disc injury model of pain in the rat tail. The general health status in rats was monitored. The nociceptive behaviour in rats was performed for mechanical allodynia using von Frey test. The HA/COLII 4.5:9 hydrogel showed higher swelling capacity than weight ratio 1:9, suggesting that a higher amount of HA can absorb a large amount of water. Both HA/COLII 4.5:9 and 1:9 hydrogel formulations had a similar degradation profile, stable to the hydrolytic process. The hWJ-MSC-encapsulated on hydrogel marked higher cell viability with round morphology shape of cells in vitro. The surgically induced disc injury in the rat tail evoked mechanical allodynia, without affecting general health status in rats. The implantation of HA/COLII 1:9 hydrogel was observed to slightly alleviate injury-induced mechanical allodynia. Fine-tuning HA/COLII-based hydrogel provides the optimal swelling capacity, stability, degradability, and non-cytotoxic, mimicking the 3D NP niche in guiding hWJ-MSCs towards NP phenotype. The HA/COLII hydrogel could be employed as an advanced cell delivery system in facilitating stem cell transplantation for intervertebral disc regeneration targeting pain

Extracellular matrix (ECM) mechanical cues guide healing in tendons. Yet, the molecular mechanisms orchestrating the healing processes remain elusive. Appropriate tissue tension is essential for tendon homeostasis and tissue health. By mapping the attainment of tensional homeostasis, we aim to understand how ECM tension regulates healing. We hypothesize that diseased tendon returns to homeostasis only after the cells reach a mechanically gated exit from wound healing. We engineered a 3D mechano-culture system to create tendon-like constructs by embedding patient-derived tendon cells into a collagen I hydrogel. Casting the hydrogel between posts anchored in silicone allowed adjusting the post stiffness. Under this static mechanical stimulation, cells remodel the (unorganized) collagen representing wound healing mechanisms. We quantified tissue-level forces using post deflection measurements. Secreted ECM was visualized by metabolic labelling with non-canonical amino acids, click chemistry and confocal microscopy. We blocked cell-mediated actin-myosin contractility using a ROCK inhibitor (Y27632) to explore the involvement of the Rho/ROCK pathway in tension regulation. Tissue tension forces reached the same homeostatic level at day 21 independent of post compliance (p = 0.9456). While minimal matrix was synthesized in early phases of tissue formation (d3-d5), cell-deposited ECM was present in later stages (d7-d9). More ECM was deposited by tendon constructs cultured on compliant (1Nm) compared to rigid posts (p = 0.0017). Matrix synthesized by constructs cultured on compliant posts was less aligned (greater fiber dispersion, p = 0.0021). ROCK inhibition significantly decreased tissue-level tensional forces (p < 0.0001). Our results indicate that tendon cells balance matrix remodeling and synthesis during tissue repair to reach an intrinsically defined “mechanostat setpoint” guiding tension-mediated exit from wound healing towards homeostasis. We are identifying specific molecular mechanosensors governing tension-regulated healing in tendon and investigate the Rho/ROCK system as their possible downstream pathway

Collagen and hyaluronic acid are two major components of intervertebral disc (IVD). They give resistance and hydration to Nucleus Pulposus. In this study, we assessed the impact of Collagen (COLL) and Hyaluronic acid-Tyramine (THA) contents on the mechanical properties and the structure of composite hydrogels. For this purpose, a range of composites were obtained using a 4 mg/mL collagen concentration and different COLL/THA ratios from 8:1 to 1:5 (w/w). Composite gelling was performed by pH increase, triggering collagen fibrillogenesis and oxidative coupling of tyramine moieties in THA catalyzed by H. 2. O. 2. and horseradish peroxidase (HRP). To modulate the THA gelling kinetic, different HRP concentrations (0.05; 0.1 and 0.5 U/mL) were used. Composites with a low THA content exhibited a fibrillar structure and possessed mechanical properties close to those of pure collagen hydrogels (200 Pa). From the ratio 1:1, the storage modulus increased to reach c.a 1200 Pa for the ratio 1:5. From the ratio 1:2, the fibrillar structure disappeared and sheets, characteristic of THA hydrogels, were observed. The HRP activity dramatically impacted the physical properties. A rapid THA gelling associated with a high THA content tended to destabilize collagen fibrils and promoted the formation of covalent bond between collagen and THA. On the opposite a slow gelling kinetic favored collagen fibril formation up to the COLL/THA ratio 1:2. Taken together, these results show that a slow gelling and an 8 mg/mL THA concentration are the appropriate conditions to obtain biomimetic biomaterials for the treatment of Nucleus Pulposus

Control of stem cell fate and function is critical for clinical and academic work. By combining surface chemistry-driven extracellular matrix (ECM) assembly with mesenchymal stem cells (MSCs) we are developing a system which can be used to regulate the behaviour of MSCs. The conformation of the ECM glycoprotein fibronectin (Fn) is different when adsorbed onto poly methylacrylate (PMA) where it is globular, and on poly ethylacrylate (PEA) where it forms a physiologically-similar network. [1]. (Fig. 1). Using these polymers to govern Fn conformation, we are developing a 3D system incorporating MSC-responsive growth factors (GFs) and bone marrow MSCs capable of regulating MSC behaviour. Toluene-dissolved PMA and PEA were spin coated onto glass coverslips before solvent extraction in vacuo and UV sterilisation. 20 mg ml. −1. human plasma FN was adsorbed onto the surfaces followed by 25 ng ml. −1. recombinant human BMP2/VEGF. FN conformations were characterised by atomic force microscopy (AFM). A collagen hydrogel was placed above the substrate. Adult human bone marrow STRO-1+ were cultured on the substrates for 3 weeks in supplemented DMEM. Expression of MSC stemness and HSC maintenance factors were analysed by In-Cell Western assay. To establish the best combination of polymer/FN/GF, MSC stemness markers (ALCAM, NESTIN and STRO1), osteogenic differentiation markers (OCN and OPN) and bone marrow markers (SCF and VCAM1) were measured in MSCs cultured for 3-weeks on substrates. OCN, SCF, and VCAM1 expression was enhanced across all combinations compared to glass control, while for ALCAM/STRO1/NESTIN and OPN, PEA combinations enhanced their expression. PEA + FN + VEGF appeared to be system best suited to maintaining MSC stemness and supporting expression of osteogenesis markers and bone marrow markers. We have shown that MSCs maintain their stem cells state and express high levels of SCF and VCAM-1 when cultured on PEA with adsorbed Fn and VEGF or BMP2. Next stages of this work will use PCR to verify results and analyse expression of other MSC markers to develop a role for these synthetic polymers as biomaterials

To prevent infections after orthopedic surgery, intravenous antibiotics are administered perioperatively. Cefazolin is widely used as the prophylactic antibiotic of choice. Systemic antibiotic therapy may however be less effective in longstanding surgery where bone allografts are used. Bone chips have been shown to be an effective carrier for certain types of antibiotics. Bone allografts impregnated with antibiotics may therefore provide the necessary local antibiotic levels for prophylaxis. To be efficient, a prolonged release from these bonechips is required. In contrast to vancomycin, for which prolonged release has clearly been proven effective from Osteomycin®, a commercially available impregnated bone allograft, no prolonged release bone chip preparations have been described so far for cefazolin. We developed a protocol to bind cefazolin in the porous structure of bone chips by means of a hydrogel composed of proteins naturally present in the human body. Three types of bone chips were evaluated: fresh frozen, decellularized frozen and decellularized lyophilized. Bone chips were incubated with 20 mg/ml cefazolin or treated with liquid hydrogel containing either 1 mg/ml fibrin or 1 mg/ml collagen and 20 mg/ml cefazolin. The cefazolin hydrogel was distributed in the porous structure by short vacuum treatment. Bone chips with cefazolin but without hydrogel were either incubated for 20 min- 4h or also treated with vacuum. Cefazolin elution of bone chips was carried out in fetal bovine serum and analyzed by Ultra Performance Liquid Chromatography – Diode Array Detection. Soaking of bone chips without hydrogel resulted in a quick release of cefazolin, which was limited to 4 hours. When vacuum was applied elution of >1 µg/ml cefazolin was measured for up to 36 hours. Combination with collagen hydrogel resulted in a higher cefazolin concentration released at 24 hours (3.9 vs 0.3 µg/ml), but not in a prolonged release. However, combination of decellularized frozen bone chips with fibrin hydrogel resulted in an initial release of 533 µg/ml followed by a gradual decline reaching the minimal inhibitory concentration for S. aureus at 72 hours (1.7 µg/ml), while not measurable anymore after 92 hours. Processed bone chips with hydrogel-cefazolin showed a markedly prolonged cefazolin release. When combined with a fibrin hydrogel, high initial peak levels of cefazolin were obtained, followed by a decreasing release over the following three days. This elution profile is desirable, since high initial levels are important to maximize anti-bacterial action whereas low levels of antibiotic for a limited time may stimulate osteogenesis. It is important that antibiotic release is ending after a few days as prolonged low levels of antibiotics are not clinically helpful and may lead to antibiotic resistance. Further preclinical studies are warranted to show effectiveness of hydrogel-cefazolin impregnated bone chips

The gelatin-based haemostyptic compound Spongostan was tested as a three-dimensional (3D) chondrocyte matrix in an in vitro model for autologous chondrocyte transplantation using cells harvested from bovine knees. In a control experiment of monolayer cultures, the proliferation or de-differentiation of bovine chondrocytes was either not or only marginally influenced by the presence of Spongostan (0.3 mg/ml).

In monolayers and 3-D Minusheet culture chambers, the cartilage-specific differentiation markers aggrecan and type-II collagen were ubiquitously present in a cell-associated fashion and in the pericellular matrix. The Minusheet cultures usually showed a markedly higher mRNA expression than monolayer cultures irrespective of whether Spongostan had been present or not during culture. Although the de-differentiation marker type-I collagen was also present, the ratio of type-I to type-II collagen or aggrecan to type-I collagen remained higher in Minusheet 3-D cultures than in monolayer cultures irrespective of whether Spongostan had been included in or excluded from the monolayer cultures. The concentration of GAG in Minusheet cultures reached its maximum after 14 days with a mean of 0.83 ± 0.8 μg/10⁶ cells; mean ±, sem, but remained considerably lower than in monolayer cultures with/without Spongostan.

Our results suggest that Spongostan is in principle suitable as a 3-D chondrocyte matrix, as demonstrated in Minusheet chambers, in particular for a culture period of 14 days. Clinically, differentiating effects on chondrocytes, simple handling and optimal formability may render Spongostan an attractive 3-D scaffold for autologous chondrocyte transplantation.