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Chondrogenically primed mesenchymal stem cell-seeded alginate hydrogels promote early bone formation in critically-sized defects
Accepted Manuscript Chondrogenically Primed Mesenchymal Stem Cell-Seeded Alginate Hydrogels Promote Early Bone Formation in Critically-Sized Defects G.M. Cunniffe, T. Vinardell, E.M. Thompson, A. Daly, A. Matsiko, F.J. O’Brien, D.J. Kelly PII: DOI: Reference:
S0014-3057(15)00369-9 http://dx.doi.org/10.1016/j.eurpolymj.2015.07.021 EPJ 6987
To appear in:
European Polymer Journal
Received Date: Revised Date: Accepted Date:
21 January 2015 5 July 2015 9 July 2015
Please cite this article as: Cunniffe, G.M., Vinardell, T., Thompson, E.M., Daly, A., Matsiko, A., O’Brien, F.J., Kelly, D.J., Chondrogenically Primed Mesenchymal Stem Cell-Seeded Alginate Hydrogels Promote Early Bone Formation in Critically-Sized Defects, European Polymer Journal (2015), doi: http://dx.doi.org/10.1016/ j.eurpolymj.2015.07.021
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Title: Chondrogenically Primed Mesenchymal Stem Cell-Seeded Alginate Hydrogels Promote Early Bone Formation in Critically-Sized Defects Authors: G. M. Cunniffe1,2,3, T. Vinardell4, E. M. Thompson1,5, A. Daly1,2,3, A. Matsiko1,5, F. J. O’Brien1,2,3,5, D. J. Kelly1,2,3* Affiliations: 1
Trinity Centre for Bioengineering, Trinity Biomedical Sciences Institute, Trinity College
Dublin, Dublin 2, Ireland. 2
Department of Mechanical and Manufacturing Engineering, School of Engineering, Trinity
College Dublin, Dublin 2, Ireland. 3
Advanced Materials and Bioengineering Research Centre, Trinity College Dublin & RCSI,
Dublin 2, Ireland. 4
School of Agriculture and Food Science, University College Dublin, Belfield, Dublin 4,
Ireland. 5
Tissue Engineering Research Group, Dept. of Anatomy, Royal College of Surgeons in
Ireland, Dublin 2, Ireland *Corresponding author: Daniel J. Kelly, Ph.D. Address: Department of Mechanical and Manufacturing Engineering, School of Engineering, Parson’s Building Trinity College Dublin, Dublin 2, Ireland Telephone: +353-1-896-3947 E-mail address: [email protected]
Key Words: Alginate, Large Bone Regeneration, Degradation, Endochondral Ossification,
Abstract: Hypertrophic cartilaginous grafts can be engineered in vitro using bone marrow derived Mesenchymal Stem Cells (MSCs). When such engineered tissues are implanted in vivo they have been shown to induce bone formation by recapitulating aspects of the developmental process of endochondral ossification. Alginate, a naturally sourced and biocompatible hydrogel, offers an attractive 3D environment to facilitate the in vitro chondrogenesis of MSCs. Furthermore, such alginate hydrogels can potentially be used to engineer cartilage tissues of scale to promote endochondral bone regeneration in large bone defects. The aim of this study was to investigate the ability of chondrogenically-primed MSC-laden alginate hydrogels to induce healing in two distinct critically-sized defect models. Bone marrow derived MSCs were seeded into alginate hydrogels, chondrogenically primed in vitro in the presence of TGF-β3 and then implanted into either a critically-sized rat cranial or femoral defect. µCT analysis 4 weeks post-implantation revealed significantly higher levels of mineralization within the femoral defects treated with MSC-laden alginate hydrogels compared to untreated empty controls, with similar results observed within the cranial defects. However, any newly deposited bone was generated appositional to the alginate material, and occurred only superficially or where the alginate was seen to degrade. Alginate material was found to persist within both orthotopic locations 8 weeks post-implantation, with its slow rate of degradation appearing to prevent complete bone regeneration. In conclusion, while chondrogenically primed MSC-alginate constructs can act as templates to treat critically-sized defects within bones formed through either intramembranous or endochondral ossification, further optimization of the degradation kinetics of the hydrogel itself will be required to accelerate bone tissue deposition and facilitate complete regeneration of such defects.
Main Text:
1. Introduction: Critically-sized bone defects, beyond the self-regenerating ability of the body, are commonly occurring clinical problems which may arise from issues such as trauma, bone disease and cancer. The repair of such defects typically relies on the use of bone grafting, whereby bone can be sourced from within the patient’s own body (an autograft), or from donated bone (an allograft). Both approaches have associated limitations, including the lack of available autograft bone, the additional risk, pain and trauma due to harvesting this bone, and the risk of an immune rejection of donated tissue. Tissue engineering or regenerative medicine aims to provide an alternative source of grafting material, by engineering a suitable tissue via the combination of scaffolding materials, cells and bioactive signals in vitro which can then be implanted in vivo to support or enhance the self-healing process of the body [1, 2]. Initial attempts to engineer such tissues focused on the direct generation of a bonelike material by inducing osteogenesis of mesenchymal stem cells (MSCs) or other osteoprogenitor cells; a strategy resembling the developmental process of intramembranous ossification. However some problems have arisen following implantation of such tissues, including a lack of vasculature in-growth, core degradation and necrosis due to a lack of available nutrients. This may be attributed to the establishment of a densely mineralized matrix within these constructs prior to implantation which may act to inhibit the ingrowth of host vessels [3, 4]. More recently, efforts have turned to engineering a cartilage-like tissue in vitro in an attempt to recapitulate the process of endochondral ossification, whereby a cartilage template is converted to mature bone. This is the developmental process through which long bones form, and is the typical healing response observed during long bone
fracture repair [5, 6]. During endochondral ossification, chondrocytes in a cartilage rudiment undergo hypertrophy and begin to secrete signals which induce the ingrowth of a vasculature network and the mineralization of the surrounding matrix. Therefore, the implantation of tissue engineered cartilage is conceived as a novel strategy to promote endochondral ossification within critically-sized defects, eventually leading to the establishment of a vascularized, mineralized and mechanically functional extracellular matrix [7]. The potential of this approach has been demonstrated using MSC-derived engineered cartilaginous grafts in both ectopic (subcutaneous) environments and within orthotopic defects [8-14]. Challenging orthotopic defects often require treatment with a construct of specific and large dimensions, which will have to be engineered and cultured in vitro prior to implantation [15]. The use of a supporting scaffold or hydrogel can facilitate the scaling-up of such engineered constructs to clinically relevant sizes [16-22], and the choice of a suitable 3D material is vital to ensure success. Appropriate biomaterials should not elicit a rejectory immune response, should provide sufficient mechanical support to allow for surgical implantation, should induce a favorable reaction from implanted or host cells, and it should degrade at a suitable rate to be replaced by newly forming tissue. Therefore identification of a suitable scaffolding material to tissue engineer scaled-up cartilage will be central to the successful realization of endochondral bone tissue engineering strategies. Alginate is a naturally derived hydrogel material which offers many advantages for use in such tissue engineering applications including excellent biocompatibility and ease of gelation to form constructs with specific dimensions [23-25]. Robust chondrogenesis of MSCs has been achieved using alginate hydrogels [26-28], and it has also been shown to act as a suitable template for ectopic bone growth [29, 30]. This hydrogel has also been successfully used as a growth factor delivery system to facilitate regeneration of bone defects [31, 32].
We have recently demonstrated that cartilaginous tissues engineered using MSCladen alginate hydrogels promote ectopic bone formation following subcutaneous implantation into nude mice [30][33]; however the capacity of such constructs to accelerate the regeneration of critically-sized orthotopic bone defects has yet to be assessed. The objective of this proof of concept study was therefore to determine the efficacy of MSC-laden alginate gels to undergo chondrogenesis in vitro and induce osteogenesis within criticallysized cranial and femoral bone defects in vivo. These two models were selected to identify any differential response of healing within bones which formed through different pathways during skeletogenesis, with the cranium developing through the intramembranous pathway and the femur forming by endochondral ossification. To facilitate vascularization and mineralization of these constructs in vivo, the architecture of the alginate hydrogels was also modified to include channels which have previously been shown to accelerate mineralization of engineered cartilage grafts in ectopic locations [34]. De novo bone formation was investigated using microCT to identify and quantify mineralization, and histological analysis was used to evaluate the nature of the repair tissue within untreated and treated defects.
2. Materials and Methods: 2.1 Isolation and expansion of MSCs Bone marrow derived MSCs were isolated from the femoral shaft of Fischer rats and expanded in expansion medium (high glucose Dulbecco’s modified eagle’s medium GlutaMAX (hgDMEM) supplemented with 10% v/v foetal bovine serum (FBS), 100 U/mL penicillin – 100 µg/mL streptomycin (all Gibco, Biosciences, Dublin Ireland), 2.5 µg/mL amphotericin B (Sigma-Aldrich, Dublin, Ireland) and 5 ng/mL human fibroblastic growth factor-2 (FGF-2; Prospec-Tany TechnoGene Ltd., Israel) and expanded to passage 2 at 20% pO2.
2.2 MSC-laden Alginate hydrogels Alginate constructs were fabricated by placing MSC-laden alginate (2% w/v ultrapure, low viscosity (20-200 mPas) sodium alginate; Pronova, FMC Biopolymer, Norway) into custom designed PDMS moulds and allowing gelation (30 mins- cranial mould, 45mins- femoral mould) to occur by covering the alginate solution in the moulds with a solid 4% agarose/50 mM CaCl2 slab. MSCs were trypsinized, counted and re-suspended into a single-cell solution in the 2% alginate (2x107 MSCs/mL) prior to gelation. Two moulds were designed to yield final gels with dimensions (i) 7mm diameter x 2mm height for the cranial defect, and (ii) 4mm diameter x 5mm height for the femoral defect. Channels were introduced into the alginate via the PDMS mould which contained an array of pillars (each 500 µm in diameter) and ran in the longitudinal direction in the cranial mould and the transverse direction in the femoral mould. Both gels were cultured in vitro for 4 weeks in chondrogenic medium followed by 3 weeks in hypertrophic medium Chondrogenic medium contained hgDMEM GlutaMAX supplemented with 100 U/mL penicillin/streptomycin (both Gibco), 100 μg/mL sodium pyruvate, 40 μg/mL L-proline, 50 μg/mL L-ascorbic acid-2-phosphate, 4.7 μg/mL linoleic acid, 1.5 mg/mL bovine serum albumin, 1×insulin–transferrin–selenium, 100 nM dexamethasone (all from Sigma Aldrich), 2.5 µg/mL amphotericin B, and 10 ng/mL of human transforming growth factor-β3 (TGF-β3) (Prospec-Tany TechnoGene Ltd., Israel) at 5% pO2. The hypertrophic medium consisted of hgDMEM GlutaMAX supplemented with 100 U/mL penicillin/streptomycin, 100 μg/mL sodium pyruvate, 40 μg/mL L-proline, 50 μg/mL L-ascorbic acid-2-phosphate, 4.7 μg/mL linoleic acid, 1.5 mg/mL bovine serum albumin, 1×insulin–transferrin–selenium, 1 nM dexamethasone, 2.5 µg/mL amphotericin B, 1 nM L-thyroxine (Sigma-Aldrich) and 20 µg/mL β-GP at 20% pO2.
2.3 Live/dead confocal microscopy Cell viability within the alginate constructs was assessed after 7 weeks of in vitro culture using a LIVE/DEAD® viability/cytotoxicity assay kit (Invitrogen, Bio-science, Ireland). Briefly, constructs were cut in half, washed in PBS followed by incubation in PBS containing 2 μM calcein AM (green fluorescence of membrane for live cells) and 4 μM ethidium homodimer-1 (red fluorescence of DNA for dead cells; both from Cambridge Bioscience, UK). Sections were again washed in PBS, imaged at magnification ×10 with an Olympus FV-1000 Point-Scanning Confocal Microscope (Southend-on-Sea, UK) at 515 and 615 nm channels and analysed using FV10-ASW 2.0 Viewer software.
2.4 Histological and immunohistochemical analysis Constructs were fixed in 4% paraformaldehyde, dehydrated in a graded ethanol series, embedded in paraffin wax, sectioned at 8 μm and affixed to microscope slides. Postimplantation constructs were decalcified in EDTA for up to 2 weeks. The sections were stained with haematoxylin and eosin (H&E) to assess bone formation, and aldehyde fuchsin/ alcian blue to assess sGAG content. Histomorphometric analysis was performed on H&E stained slices, with areas of new bone formation identified by deep pink staining and quantified by measuring their area in each section (Image J program) and calculating the mean total area per group (mm^2).
2.5 Femoral defect implantation A 5 mm weight-bearing segmental femoral defect was created in adult male Fischer rats following an established procedure [35]. Ethical approval was given by the Research Ethics Committee of the Royal College of Surgeons in Ireland. Anaesthesia was induced with an
intraperitoneal injection of xylazine hydrochloride (10 mg/kg) and ketamine hydrochloride (75 mg/kg), and maintained with inhalational isoflurane and oxygen (0.5-2% isoflurane). MSC-alginate hydrogels were press-fit into the defect site, and repair tissue was harvested for analysis at 4 and 8 weeks post-implantation (n=4). Briefly, the shaft of the right femur was exposed by dissections and the periosteum was scraped back to allow access to the bone. A weight-bearing polyetheretherketone (PEEK) internal fixation plate was secured to the exposed femur with four screws into pre-drilled holes. A 5 mm mid-diaphyseal defect was then created using a dental drill fitted with 2 small circular parallel saw blades welded to a narrow straight rod separated with a 5 mm spacer. The defect site was thoroughly irrigated with saline to remove bone debris before it was treated with the MSC-alginate gels. Atipamezole hydrochloride was given to reverse sedation. Pain relief was administered via carprofen and buprenorphine subcutaneous injections over the following 2 days. On dates of scheduled explant retrieval, rats were sacrificed by CO2 asphyxiation and cervical dislocation. After 4 and 8 weeks, the repaired femur, with the PEEK plate fixator intact, was carefully separated from the adjacent hip and knee joints for analysis.
2.6 Cranial defect implantation A 7 mm cranial defect was created in adult male Fischer rats following an established procedure [3] and the same anaesthesia and pain relief protocol as outlined in the previous section, with n=4 animals per group at two time points of 4 and 8 weeks. A 7 mm circular transosseous defect was created on the left side of the rat parietal calvarium using a grinding burr and the cranial MSC-alginate hydrogel was immediately implanted into the defect or left untreated for the empty defect groups before closing of the wound with sutures. Calvaria were harvested for evaluation 4 and 8 weeks post-implantation.
2.7 Micro-computed tomography Micro-computed tomography (µCT) scans were carried out on the explants from the cranial and femoral defect studies in order to visualise and quantify mineral content. A Scanco Medical 40 μCT system (Scanco Medical, Bassersdorf, Switzerland) was used for evaluation with a 70 kVp X-ray source at 114 μA. Four constructs were analysed per experimental group. Reconstructed 3D images were generated from the scans and used to visualise mineral distribution throughout the implanted scaffold or control group. Quantification was performed by setting a threshold of 210 (corresponding to a density of 399.5 mg hydroxyapatite/cm3) and recording the bone volume (BV) within the defect per total volume (BV/TV), using a consistent total volume from sample to sample which eliminated original bone from the calculations.
2.8 Statistical Analysis Statistical analysis was carried out using GraphPad software. The results are reported as means ± standard deviation and groups were analysed using Student’s two-tailed t-tests or by a general linear model for analysis of variance with groups of factors. Tukey’s post-hoc test was used to compare conditions. Significance was accepted at a level of p < 0.05.
3. Results and Discussion
3.1 Scaled up cartilaginous grafts can be engineered using channelled MSC-laden alginate hydrogels Analysis of MSC-laden alginate hydrogels (Fig. 1A) following 7 weeks in vitro demonstrated that the majority of MSCs remained viable (Fig. 1B), and that they had undergone chondrogenesis, depositing a cartilage-like matrix rich in sulfated glycosaminoglycans
(sGAG; Fig. 1C). These cartilaginous grafts were then implanted into either cranial or femoral bone defects (Fig. 1D). No evidence of mineralization was detected in the engineered tissue prior to implantation using µCT analysis at a threshold of 210. There appeared to be more cell death evident in the central region of the MSC-alginate hydrogel engineered for the femoral defect, which may be due to the larger dimensions of this gel, with a greater axial distance for nutrients and oxygen to travel to the cells in the centre. Alternatively it may be due to the longer cross-linking time required for complete gelation to occur when the gel was initially formed.
Fig. 1A. Macroscopic image of MSC-laden alginate gels following 7 weeks in vitro. (B) Confocal microscopy images of gels after 7 weeks of culture, demonstrating that a majority of cells remained viable, (C) homogeneous deposition of sGAG achieved in the channelled gels shown by Alcian blue staining, and (D) the gels in situ in their respective defect models
3.2 Chondrogenically primed MSC-laden alginate hydrogels promote early bone formation in critically-sized femoral defects
Matrix mineralization and neo-bone formation within the femoral defect was quantified using µCT analysis, with significantly greater levels of mineralization observed following treatment with cartilaginous tissues engineered using MSC-laden alginate hydrogels after 4 weeks compared to untreated empty defect controls (Fig. 2). This trend continued at week 8, although at this time-point no significant differences in mineral deposition were observed between treated and untreated defects. The reconstructed images revealed a non-homogenous distribution of the mineralized tissue within the alginate treated groups, with mineralized sections appearing to be confined to narrow sections occurring randomly across the length of the defect. No obvious correlation was observed between the pattern of bone formation and the geometry of the channels running transversely through the hydrogels.
Fig. 2A. Reconstructed μCT images of femoral defects left untreated at 4 and 8 weeks and (B) treated with chondrogenically primed MSC-alginate gels. (C) Bone volume per total volume (BV/TV) within the defect site for untreated and treated groups
Histological analysis of the repair tissue generated following implantation of cartilaginous constructs revealed a large quantity of residual alginate remaining within the defect site at both 4 and 8 weeks (Fig. 3). H&E staining detected the formation of de novo bone forming along the surface of the alginate material; however despite a trend towards higher levels of bone, no significant difference was detected between groups via histomorphometric analysis at 8 weeks (Fig. 3D). This appositional bone growth may explain the unusual distribution of newly mineralized matrix observed in the μCT scans, as new tissue may only be permitted to form in superficial regions or where the alginate has begun to degrade. Contrary to this, it is clear that a fibrous tissue formed across the defect in the untreated controls, with no evidence of significant de novo bone formation. The finding that such cartilaginous constructs provided a platform for osteogenesis is in agreement with previous studies that have demonstrated that tissue engineered hyaline cartilaginous grafts provided a substrate capable of supporting both osteoblast and hMSC maturation and osteogenesis in vitro and in vivo following subcutaneous implantation into nude mice [36]. In the treated groups, it is likely that residual alginate within the defect is hindering vascularization and the deposition of new bone tissue. Indeed, no significant difference in blood vessel number was observed in empty and treated defects (data not shown). This residual alginate may also potentially explain why the higher levels of mineralization and de novo bone formation observed 4 weeks post-implantation in the treated groups did not persist at week 8. This stability of the alginate material in vivo suggests that the calcium mediated cross-links formed between the G blocks of the alginate monomers in vitro remain intact during this period, with additional cross-linking and/or re-cross-linking also potentially occurring in vivo due to the presence of soluble calcium ions within the mineralizing tissue. The presence of engineered cartilage within the hydrogel may also be slowing its rate of degradation in vivo. These findings are somewhat in contrast to our previous studies where
chondrogenically primed MSC-laden alginate hydrogels were implanted subcutaneously into nude mice. In that study the constructs underwent more noticeable levels of degradation at comparable timepoints, facilitating vascularization and bone formation throughout the body of the implant [30]. This points to the importance of evaluating endochondral tissue engineering strategies in orthotopic locations.
Fig. 3A. Histological images of femoral defects left untreated at 4 and 8 weeks and (B) treated with chondrogenically primed MSC-laden alginate hydrogels. (C) Higher magnification (10x) images demonstrate the appositional bone growth occurring in between the slowly degrading alginate. (D) Histomorphometric analysis of bone area within defect site 8 weeks post implantation
3.3 Engineered cartilaginous tissues can promote bone formation in critically-sized intramembranous bone defects
Engineered cartilage grafts also appeared to support mineralization within critically-sized rat cranial defects (Fig. 4). A trend towards higher levels of mineralization was observed in treated groups at both 4 weeks (p=0.1704) and 8 weeks (p=0.0671), although these differences were not statistically significant at either time point. As with the femoral defects, the location of the mineralized tissue again appeared non-uniform, with densely mineralized areas separated by large un-mineralized regions.
Fig. 4A Reconstructed μCT images of cranial defects left untreated at 4 and 8 weeks and (B) treated with chondrogenically primed MSC-laden alginate gels. (C) Bone volume per total volume (BV/TV) within the cranial defect site for untreated and alginate-treated groups
Histological analysis of the healing within the cranial defects demonstrated that similar to the femoral defect, significant appositional bone formation was observed on the surface of the MSC-laden alginate hydrogels at both 4 and 8 weeks (Fig. 5). Following quantification, a significant increase in bone area was detected in the alginate treated group when compared to the untreated within this defect model (Fig. 5D). Previous studies that have implanted
cartilaginous tissues engineered using chitosan sponges have similarly observed surface bone formation 1 month after subcutaneous implantation in nude mice, with vascularization and bone formation proceeding into the body of the implant over a 5 month period [37]. Again, the slow rate of alginate degradation appeared to hamper the widespread deposition of de novo bone, although it is clear that the chondrogenically primed MSC-laden alginate hydrogel again provided a promising template for bone growth, this time within a defect in a bone formed via intramembranous ossification.
Fig. 5A. Histological images of cranial defects left untreated at 4 and 8 weeks and (B) treated with alginate gels containing chondrogenically primed MSCs. (C) Higher magnification (20x) images demonstrate the appositional bone growth occurring along the alginate surface. (D) Histomorphometric analysis of bone area within defect site 8 weeks post implantation
In addition to providing a suitable substrate for bone formation, it is also likely that the trophic factors secreted by the hypertrophic chondrocytes within the alginate material contributed towards this appositional de novo bone. For example, positive staining for VEGF was detected pericellularly up to 8 weeks post implantation, although the overall number of blood vessels was similar in empty and treated defects in both the femoral and cranial models (data not shown). A recent study by Bourgine et al. (2014) demonstrated that factors such as BMP-2, VEGF and MMP-13 are expressed by hypertrophic chondrocytes derived from bone marrow derived MSCs, all of which would influence subsequent bone formation [38]. Alginate has been well documented as a hydrogel with excellent biocompatibility, readily processable for various applications including drug delivery and cell carriers, and it offers many options to yield tunable degradation kinetics [24, 39-41]. As previously shown following ectopic implantation of MSC-laden alginate hydrogels, the degradation rate of the material is a critical determinant of the rate of tissue formation [32, 42, 43]. Large quantities of residual alginate were still evident after 8 weeks in vivo within both the cranial and femoral defects in the present study, demonstrating the slow rate at which the alginate breaks down in vivo. This would clearly hinder vascularization of the engineered construct and its capacity to undergo endochondral ossification. As the first use, to the best of our knowledge, of chondrogenically primed MSC-alginate constructs to attempt osteogenesis via endochondral ossification in an orthotopic location, this proof of concept study provides a baseline for such applications, and suggests that tailoring the degradation rate of the material is important for facilitating endochondral bone regeneration. Rather than striving for a densely cross-linked, mechanically robust alginate material, it would appear that the target should be generate a construct with mechanical properties sufficient to withstand the extended in vitro culture period required to generate anatomically accurate, tissue engineered cartilaginous grafts in addition to surgical handling during subsequent implantation, but
which can then degrade rapidly in vivo. An alternative strategy would be to remove or uncrosslink the hydrogel once a sufficient amount of cartilaginous matrix has been deposited in vitro. Future studies in our lab will focus on various strategies to alter the degradation kinetics of the alginate material to accelerate endochondral bone formation, for instance by using gamma-irradiated, lower molecular weight alginate or partially oxidized alginate as this is known to increase the rate of degradation [24, 39, 42, 44]. Other approaches could include the incorporation of a more rapidly degrading second network within the alginate construct, the inclusion of a larger network of channels, the addition of inflammatory cytokines to enhance resorption, the chemical removal of the alginate phase entirely prior to implantation, or a reduction in the gelation time [36, 41, 45, 46]. As noted above, care needs to be taken to strike the correct balance between improving the degradation kinetics whilst ensuring the construct can remain intact during the in vitro culture phase and during surgical implantation.
4. Conclusions Scaling up of tissue engineering strategies to treat large bone defects remains a significant challenge in the field of orthopedic regenerative medicine. This study established the capability of alginate hydrogel material to support the in vitro chondrogenesis of MSCs and the subsequent initiation of neo-bone formation within large bone defects. Taken together, these results illustrate the capacity of chondrogenically primed MSC-alginate hydrogels to induce a similar healing response within critically-sized orthotopic bone defects of distinct embryological developmental pathways - the femur and the cranium. In both cases the chondrogenically primed MSC-alginate constructs appeared to actively support the deposition of new de novo bone appositionally along the surface of the engineered construct, and within regions of the implant that had degraded. Future work will focus on optimizing the
degradation kinetics of the hydrogel itself to accelerate de novo bone formation and facilitate complete regeneration of such defects.
Acknowledgements: This work was funded by the AO foundation under the large bone defect healing program. Surgical advice, PEEK fixator plates and custom designed surgical instruments were kindly provided by Professor Steve Goldstein, University of Michigan. Human bone marrow-derived MSCs were kindly provided by REMEDI, National Centre for Biomedical Engineering Science, National University of Ireland Galway, Galway, Ireland and funded by Science Foundation Ireland (grant number 09/SRC/B1794).
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Graphical abstract
Highlights: Alginate supports chondrogenesis of Mesenchymal Stem Cells Higher levels of mineralization observed in defects treated with engineered tissues Bone formation occurs appositional to the slowly degrading alginate hydrogel Hydrogel degradation rate regulates de novo bone deposition
S0014-3057(15)00369-9 http://dx.doi.org/10.1016/j.eurpolymj.2015.07.021 EPJ 6987
To appear in:
European Polymer Journal
Received Date: Revised Date: Accepted Date:
21 January 2015 5 July 2015 9 July 2015
Please cite this article as: Cunniffe, G.M., Vinardell, T., Thompson, E.M., Daly, A., Matsiko, A., O’Brien, F.J., Kelly, D.J., Chondrogenically Primed Mesenchymal Stem Cell-Seeded Alginate Hydrogels Promote Early Bone Formation in Critically-Sized Defects, European Polymer Journal (2015), doi: http://dx.doi.org/10.1016/ j.eurpolymj.2015.07.021
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Title: Chondrogenically Primed Mesenchymal Stem Cell-Seeded Alginate Hydrogels Promote Early Bone Formation in Critically-Sized Defects Authors: G. M. Cunniffe1,2,3, T. Vinardell4, E. M. Thompson1,5, A. Daly1,2,3, A. Matsiko1,5, F. J. O’Brien1,2,3,5, D. J. Kelly1,2,3* Affiliations: 1
Trinity Centre for Bioengineering, Trinity Biomedical Sciences Institute, Trinity College
Dublin, Dublin 2, Ireland. 2
Department of Mechanical and Manufacturing Engineering, School of Engineering, Trinity
College Dublin, Dublin 2, Ireland. 3
Advanced Materials and Bioengineering Research Centre, Trinity College Dublin & RCSI,
Dublin 2, Ireland. 4
School of Agriculture and Food Science, University College Dublin, Belfield, Dublin 4,
Ireland. 5
Tissue Engineering Research Group, Dept. of Anatomy, Royal College of Surgeons in
Ireland, Dublin 2, Ireland *Corresponding author: Daniel J. Kelly, Ph.D. Address: Department of Mechanical and Manufacturing Engineering, School of Engineering, Parson’s Building Trinity College Dublin, Dublin 2, Ireland Telephone: +353-1-896-3947 E-mail address: [email protected]
Key Words: Alginate, Large Bone Regeneration, Degradation, Endochondral Ossification,
Abstract: Hypertrophic cartilaginous grafts can be engineered in vitro using bone marrow derived Mesenchymal Stem Cells (MSCs). When such engineered tissues are implanted in vivo they have been shown to induce bone formation by recapitulating aspects of the developmental process of endochondral ossification. Alginate, a naturally sourced and biocompatible hydrogel, offers an attractive 3D environment to facilitate the in vitro chondrogenesis of MSCs. Furthermore, such alginate hydrogels can potentially be used to engineer cartilage tissues of scale to promote endochondral bone regeneration in large bone defects. The aim of this study was to investigate the ability of chondrogenically-primed MSC-laden alginate hydrogels to induce healing in two distinct critically-sized defect models. Bone marrow derived MSCs were seeded into alginate hydrogels, chondrogenically primed in vitro in the presence of TGF-β3 and then implanted into either a critically-sized rat cranial or femoral defect. µCT analysis 4 weeks post-implantation revealed significantly higher levels of mineralization within the femoral defects treated with MSC-laden alginate hydrogels compared to untreated empty controls, with similar results observed within the cranial defects. However, any newly deposited bone was generated appositional to the alginate material, and occurred only superficially or where the alginate was seen to degrade. Alginate material was found to persist within both orthotopic locations 8 weeks post-implantation, with its slow rate of degradation appearing to prevent complete bone regeneration. In conclusion, while chondrogenically primed MSC-alginate constructs can act as templates to treat critically-sized defects within bones formed through either intramembranous or endochondral ossification, further optimization of the degradation kinetics of the hydrogel itself will be required to accelerate bone tissue deposition and facilitate complete regeneration of such defects.
Main Text:
1. Introduction: Critically-sized bone defects, beyond the self-regenerating ability of the body, are commonly occurring clinical problems which may arise from issues such as trauma, bone disease and cancer. The repair of such defects typically relies on the use of bone grafting, whereby bone can be sourced from within the patient’s own body (an autograft), or from donated bone (an allograft). Both approaches have associated limitations, including the lack of available autograft bone, the additional risk, pain and trauma due to harvesting this bone, and the risk of an immune rejection of donated tissue. Tissue engineering or regenerative medicine aims to provide an alternative source of grafting material, by engineering a suitable tissue via the combination of scaffolding materials, cells and bioactive signals in vitro which can then be implanted in vivo to support or enhance the self-healing process of the body [1, 2]. Initial attempts to engineer such tissues focused on the direct generation of a bonelike material by inducing osteogenesis of mesenchymal stem cells (MSCs) or other osteoprogenitor cells; a strategy resembling the developmental process of intramembranous ossification. However some problems have arisen following implantation of such tissues, including a lack of vasculature in-growth, core degradation and necrosis due to a lack of available nutrients. This may be attributed to the establishment of a densely mineralized matrix within these constructs prior to implantation which may act to inhibit the ingrowth of host vessels [3, 4]. More recently, efforts have turned to engineering a cartilage-like tissue in vitro in an attempt to recapitulate the process of endochondral ossification, whereby a cartilage template is converted to mature bone. This is the developmental process through which long bones form, and is the typical healing response observed during long bone
fracture repair [5, 6]. During endochondral ossification, chondrocytes in a cartilage rudiment undergo hypertrophy and begin to secrete signals which induce the ingrowth of a vasculature network and the mineralization of the surrounding matrix. Therefore, the implantation of tissue engineered cartilage is conceived as a novel strategy to promote endochondral ossification within critically-sized defects, eventually leading to the establishment of a vascularized, mineralized and mechanically functional extracellular matrix [7]. The potential of this approach has been demonstrated using MSC-derived engineered cartilaginous grafts in both ectopic (subcutaneous) environments and within orthotopic defects [8-14]. Challenging orthotopic defects often require treatment with a construct of specific and large dimensions, which will have to be engineered and cultured in vitro prior to implantation [15]. The use of a supporting scaffold or hydrogel can facilitate the scaling-up of such engineered constructs to clinically relevant sizes [16-22], and the choice of a suitable 3D material is vital to ensure success. Appropriate biomaterials should not elicit a rejectory immune response, should provide sufficient mechanical support to allow for surgical implantation, should induce a favorable reaction from implanted or host cells, and it should degrade at a suitable rate to be replaced by newly forming tissue. Therefore identification of a suitable scaffolding material to tissue engineer scaled-up cartilage will be central to the successful realization of endochondral bone tissue engineering strategies. Alginate is a naturally derived hydrogel material which offers many advantages for use in such tissue engineering applications including excellent biocompatibility and ease of gelation to form constructs with specific dimensions [23-25]. Robust chondrogenesis of MSCs has been achieved using alginate hydrogels [26-28], and it has also been shown to act as a suitable template for ectopic bone growth [29, 30]. This hydrogel has also been successfully used as a growth factor delivery system to facilitate regeneration of bone defects [31, 32].
We have recently demonstrated that cartilaginous tissues engineered using MSCladen alginate hydrogels promote ectopic bone formation following subcutaneous implantation into nude mice [30][33]; however the capacity of such constructs to accelerate the regeneration of critically-sized orthotopic bone defects has yet to be assessed. The objective of this proof of concept study was therefore to determine the efficacy of MSC-laden alginate gels to undergo chondrogenesis in vitro and induce osteogenesis within criticallysized cranial and femoral bone defects in vivo. These two models were selected to identify any differential response of healing within bones which formed through different pathways during skeletogenesis, with the cranium developing through the intramembranous pathway and the femur forming by endochondral ossification. To facilitate vascularization and mineralization of these constructs in vivo, the architecture of the alginate hydrogels was also modified to include channels which have previously been shown to accelerate mineralization of engineered cartilage grafts in ectopic locations [34]. De novo bone formation was investigated using microCT to identify and quantify mineralization, and histological analysis was used to evaluate the nature of the repair tissue within untreated and treated defects.
2. Materials and Methods: 2.1 Isolation and expansion of MSCs Bone marrow derived MSCs were isolated from the femoral shaft of Fischer rats and expanded in expansion medium (high glucose Dulbecco’s modified eagle’s medium GlutaMAX (hgDMEM) supplemented with 10% v/v foetal bovine serum (FBS), 100 U/mL penicillin – 100 µg/mL streptomycin (all Gibco, Biosciences, Dublin Ireland), 2.5 µg/mL amphotericin B (Sigma-Aldrich, Dublin, Ireland) and 5 ng/mL human fibroblastic growth factor-2 (FGF-2; Prospec-Tany TechnoGene Ltd., Israel) and expanded to passage 2 at 20% pO2.
2.2 MSC-laden Alginate hydrogels Alginate constructs were fabricated by placing MSC-laden alginate (2% w/v ultrapure, low viscosity (20-200 mPas) sodium alginate; Pronova, FMC Biopolymer, Norway) into custom designed PDMS moulds and allowing gelation (30 mins- cranial mould, 45mins- femoral mould) to occur by covering the alginate solution in the moulds with a solid 4% agarose/50 mM CaCl2 slab. MSCs were trypsinized, counted and re-suspended into a single-cell solution in the 2% alginate (2x107 MSCs/mL) prior to gelation. Two moulds were designed to yield final gels with dimensions (i) 7mm diameter x 2mm height for the cranial defect, and (ii) 4mm diameter x 5mm height for the femoral defect. Channels were introduced into the alginate via the PDMS mould which contained an array of pillars (each 500 µm in diameter) and ran in the longitudinal direction in the cranial mould and the transverse direction in the femoral mould. Both gels were cultured in vitro for 4 weeks in chondrogenic medium followed by 3 weeks in hypertrophic medium Chondrogenic medium contained hgDMEM GlutaMAX supplemented with 100 U/mL penicillin/streptomycin (both Gibco), 100 μg/mL sodium pyruvate, 40 μg/mL L-proline, 50 μg/mL L-ascorbic acid-2-phosphate, 4.7 μg/mL linoleic acid, 1.5 mg/mL bovine serum albumin, 1×insulin–transferrin–selenium, 100 nM dexamethasone (all from Sigma Aldrich), 2.5 µg/mL amphotericin B, and 10 ng/mL of human transforming growth factor-β3 (TGF-β3) (Prospec-Tany TechnoGene Ltd., Israel) at 5% pO2. The hypertrophic medium consisted of hgDMEM GlutaMAX supplemented with 100 U/mL penicillin/streptomycin, 100 μg/mL sodium pyruvate, 40 μg/mL L-proline, 50 μg/mL L-ascorbic acid-2-phosphate, 4.7 μg/mL linoleic acid, 1.5 mg/mL bovine serum albumin, 1×insulin–transferrin–selenium, 1 nM dexamethasone, 2.5 µg/mL amphotericin B, 1 nM L-thyroxine (Sigma-Aldrich) and 20 µg/mL β-GP at 20% pO2.
2.3 Live/dead confocal microscopy Cell viability within the alginate constructs was assessed after 7 weeks of in vitro culture using a LIVE/DEAD® viability/cytotoxicity assay kit (Invitrogen, Bio-science, Ireland). Briefly, constructs were cut in half, washed in PBS followed by incubation in PBS containing 2 μM calcein AM (green fluorescence of membrane for live cells) and 4 μM ethidium homodimer-1 (red fluorescence of DNA for dead cells; both from Cambridge Bioscience, UK). Sections were again washed in PBS, imaged at magnification ×10 with an Olympus FV-1000 Point-Scanning Confocal Microscope (Southend-on-Sea, UK) at 515 and 615 nm channels and analysed using FV10-ASW 2.0 Viewer software.
2.4 Histological and immunohistochemical analysis Constructs were fixed in 4% paraformaldehyde, dehydrated in a graded ethanol series, embedded in paraffin wax, sectioned at 8 μm and affixed to microscope slides. Postimplantation constructs were decalcified in EDTA for up to 2 weeks. The sections were stained with haematoxylin and eosin (H&E) to assess bone formation, and aldehyde fuchsin/ alcian blue to assess sGAG content. Histomorphometric analysis was performed on H&E stained slices, with areas of new bone formation identified by deep pink staining and quantified by measuring their area in each section (Image J program) and calculating the mean total area per group (mm^2).
2.5 Femoral defect implantation A 5 mm weight-bearing segmental femoral defect was created in adult male Fischer rats following an established procedure [35]. Ethical approval was given by the Research Ethics Committee of the Royal College of Surgeons in Ireland. Anaesthesia was induced with an
intraperitoneal injection of xylazine hydrochloride (10 mg/kg) and ketamine hydrochloride (75 mg/kg), and maintained with inhalational isoflurane and oxygen (0.5-2% isoflurane). MSC-alginate hydrogels were press-fit into the defect site, and repair tissue was harvested for analysis at 4 and 8 weeks post-implantation (n=4). Briefly, the shaft of the right femur was exposed by dissections and the periosteum was scraped back to allow access to the bone. A weight-bearing polyetheretherketone (PEEK) internal fixation plate was secured to the exposed femur with four screws into pre-drilled holes. A 5 mm mid-diaphyseal defect was then created using a dental drill fitted with 2 small circular parallel saw blades welded to a narrow straight rod separated with a 5 mm spacer. The defect site was thoroughly irrigated with saline to remove bone debris before it was treated with the MSC-alginate gels. Atipamezole hydrochloride was given to reverse sedation. Pain relief was administered via carprofen and buprenorphine subcutaneous injections over the following 2 days. On dates of scheduled explant retrieval, rats were sacrificed by CO2 asphyxiation and cervical dislocation. After 4 and 8 weeks, the repaired femur, with the PEEK plate fixator intact, was carefully separated from the adjacent hip and knee joints for analysis.
2.6 Cranial defect implantation A 7 mm cranial defect was created in adult male Fischer rats following an established procedure [3] and the same anaesthesia and pain relief protocol as outlined in the previous section, with n=4 animals per group at two time points of 4 and 8 weeks. A 7 mm circular transosseous defect was created on the left side of the rat parietal calvarium using a grinding burr and the cranial MSC-alginate hydrogel was immediately implanted into the defect or left untreated for the empty defect groups before closing of the wound with sutures. Calvaria were harvested for evaluation 4 and 8 weeks post-implantation.
2.7 Micro-computed tomography Micro-computed tomography (µCT) scans were carried out on the explants from the cranial and femoral defect studies in order to visualise and quantify mineral content. A Scanco Medical 40 μCT system (Scanco Medical, Bassersdorf, Switzerland) was used for evaluation with a 70 kVp X-ray source at 114 μA. Four constructs were analysed per experimental group. Reconstructed 3D images were generated from the scans and used to visualise mineral distribution throughout the implanted scaffold or control group. Quantification was performed by setting a threshold of 210 (corresponding to a density of 399.5 mg hydroxyapatite/cm3) and recording the bone volume (BV) within the defect per total volume (BV/TV), using a consistent total volume from sample to sample which eliminated original bone from the calculations.
2.8 Statistical Analysis Statistical analysis was carried out using GraphPad software. The results are reported as means ± standard deviation and groups were analysed using Student’s two-tailed t-tests or by a general linear model for analysis of variance with groups of factors. Tukey’s post-hoc test was used to compare conditions. Significance was accepted at a level of p < 0.05.
3. Results and Discussion
3.1 Scaled up cartilaginous grafts can be engineered using channelled MSC-laden alginate hydrogels Analysis of MSC-laden alginate hydrogels (Fig. 1A) following 7 weeks in vitro demonstrated that the majority of MSCs remained viable (Fig. 1B), and that they had undergone chondrogenesis, depositing a cartilage-like matrix rich in sulfated glycosaminoglycans
(sGAG; Fig. 1C). These cartilaginous grafts were then implanted into either cranial or femoral bone defects (Fig. 1D). No evidence of mineralization was detected in the engineered tissue prior to implantation using µCT analysis at a threshold of 210. There appeared to be more cell death evident in the central region of the MSC-alginate hydrogel engineered for the femoral defect, which may be due to the larger dimensions of this gel, with a greater axial distance for nutrients and oxygen to travel to the cells in the centre. Alternatively it may be due to the longer cross-linking time required for complete gelation to occur when the gel was initially formed.
Fig. 1A. Macroscopic image of MSC-laden alginate gels following 7 weeks in vitro. (B) Confocal microscopy images of gels after 7 weeks of culture, demonstrating that a majority of cells remained viable, (C) homogeneous deposition of sGAG achieved in the channelled gels shown by Alcian blue staining, and (D) the gels in situ in their respective defect models
3.2 Chondrogenically primed MSC-laden alginate hydrogels promote early bone formation in critically-sized femoral defects
Matrix mineralization and neo-bone formation within the femoral defect was quantified using µCT analysis, with significantly greater levels of mineralization observed following treatment with cartilaginous tissues engineered using MSC-laden alginate hydrogels after 4 weeks compared to untreated empty defect controls (Fig. 2). This trend continued at week 8, although at this time-point no significant differences in mineral deposition were observed between treated and untreated defects. The reconstructed images revealed a non-homogenous distribution of the mineralized tissue within the alginate treated groups, with mineralized sections appearing to be confined to narrow sections occurring randomly across the length of the defect. No obvious correlation was observed between the pattern of bone formation and the geometry of the channels running transversely through the hydrogels.
Fig. 2A. Reconstructed μCT images of femoral defects left untreated at 4 and 8 weeks and (B) treated with chondrogenically primed MSC-alginate gels. (C) Bone volume per total volume (BV/TV) within the defect site for untreated and treated groups
Histological analysis of the repair tissue generated following implantation of cartilaginous constructs revealed a large quantity of residual alginate remaining within the defect site at both 4 and 8 weeks (Fig. 3). H&E staining detected the formation of de novo bone forming along the surface of the alginate material; however despite a trend towards higher levels of bone, no significant difference was detected between groups via histomorphometric analysis at 8 weeks (Fig. 3D). This appositional bone growth may explain the unusual distribution of newly mineralized matrix observed in the μCT scans, as new tissue may only be permitted to form in superficial regions or where the alginate has begun to degrade. Contrary to this, it is clear that a fibrous tissue formed across the defect in the untreated controls, with no evidence of significant de novo bone formation. The finding that such cartilaginous constructs provided a platform for osteogenesis is in agreement with previous studies that have demonstrated that tissue engineered hyaline cartilaginous grafts provided a substrate capable of supporting both osteoblast and hMSC maturation and osteogenesis in vitro and in vivo following subcutaneous implantation into nude mice [36]. In the treated groups, it is likely that residual alginate within the defect is hindering vascularization and the deposition of new bone tissue. Indeed, no significant difference in blood vessel number was observed in empty and treated defects (data not shown). This residual alginate may also potentially explain why the higher levels of mineralization and de novo bone formation observed 4 weeks post-implantation in the treated groups did not persist at week 8. This stability of the alginate material in vivo suggests that the calcium mediated cross-links formed between the G blocks of the alginate monomers in vitro remain intact during this period, with additional cross-linking and/or re-cross-linking also potentially occurring in vivo due to the presence of soluble calcium ions within the mineralizing tissue. The presence of engineered cartilage within the hydrogel may also be slowing its rate of degradation in vivo. These findings are somewhat in contrast to our previous studies where
chondrogenically primed MSC-laden alginate hydrogels were implanted subcutaneously into nude mice. In that study the constructs underwent more noticeable levels of degradation at comparable timepoints, facilitating vascularization and bone formation throughout the body of the implant [30]. This points to the importance of evaluating endochondral tissue engineering strategies in orthotopic locations.
Fig. 3A. Histological images of femoral defects left untreated at 4 and 8 weeks and (B) treated with chondrogenically primed MSC-laden alginate hydrogels. (C) Higher magnification (10x) images demonstrate the appositional bone growth occurring in between the slowly degrading alginate. (D) Histomorphometric analysis of bone area within defect site 8 weeks post implantation
3.3 Engineered cartilaginous tissues can promote bone formation in critically-sized intramembranous bone defects
Engineered cartilage grafts also appeared to support mineralization within critically-sized rat cranial defects (Fig. 4). A trend towards higher levels of mineralization was observed in treated groups at both 4 weeks (p=0.1704) and 8 weeks (p=0.0671), although these differences were not statistically significant at either time point. As with the femoral defects, the location of the mineralized tissue again appeared non-uniform, with densely mineralized areas separated by large un-mineralized regions.
Fig. 4A Reconstructed μCT images of cranial defects left untreated at 4 and 8 weeks and (B) treated with chondrogenically primed MSC-laden alginate gels. (C) Bone volume per total volume (BV/TV) within the cranial defect site for untreated and alginate-treated groups
Histological analysis of the healing within the cranial defects demonstrated that similar to the femoral defect, significant appositional bone formation was observed on the surface of the MSC-laden alginate hydrogels at both 4 and 8 weeks (Fig. 5). Following quantification, a significant increase in bone area was detected in the alginate treated group when compared to the untreated within this defect model (Fig. 5D). Previous studies that have implanted
cartilaginous tissues engineered using chitosan sponges have similarly observed surface bone formation 1 month after subcutaneous implantation in nude mice, with vascularization and bone formation proceeding into the body of the implant over a 5 month period [37]. Again, the slow rate of alginate degradation appeared to hamper the widespread deposition of de novo bone, although it is clear that the chondrogenically primed MSC-laden alginate hydrogel again provided a promising template for bone growth, this time within a defect in a bone formed via intramembranous ossification.
Fig. 5A. Histological images of cranial defects left untreated at 4 and 8 weeks and (B) treated with alginate gels containing chondrogenically primed MSCs. (C) Higher magnification (20x) images demonstrate the appositional bone growth occurring along the alginate surface. (D) Histomorphometric analysis of bone area within defect site 8 weeks post implantation
In addition to providing a suitable substrate for bone formation, it is also likely that the trophic factors secreted by the hypertrophic chondrocytes within the alginate material contributed towards this appositional de novo bone. For example, positive staining for VEGF was detected pericellularly up to 8 weeks post implantation, although the overall number of blood vessels was similar in empty and treated defects in both the femoral and cranial models (data not shown). A recent study by Bourgine et al. (2014) demonstrated that factors such as BMP-2, VEGF and MMP-13 are expressed by hypertrophic chondrocytes derived from bone marrow derived MSCs, all of which would influence subsequent bone formation [38]. Alginate has been well documented as a hydrogel with excellent biocompatibility, readily processable for various applications including drug delivery and cell carriers, and it offers many options to yield tunable degradation kinetics [24, 39-41]. As previously shown following ectopic implantation of MSC-laden alginate hydrogels, the degradation rate of the material is a critical determinant of the rate of tissue formation [32, 42, 43]. Large quantities of residual alginate were still evident after 8 weeks in vivo within both the cranial and femoral defects in the present study, demonstrating the slow rate at which the alginate breaks down in vivo. This would clearly hinder vascularization of the engineered construct and its capacity to undergo endochondral ossification. As the first use, to the best of our knowledge, of chondrogenically primed MSC-alginate constructs to attempt osteogenesis via endochondral ossification in an orthotopic location, this proof of concept study provides a baseline for such applications, and suggests that tailoring the degradation rate of the material is important for facilitating endochondral bone regeneration. Rather than striving for a densely cross-linked, mechanically robust alginate material, it would appear that the target should be generate a construct with mechanical properties sufficient to withstand the extended in vitro culture period required to generate anatomically accurate, tissue engineered cartilaginous grafts in addition to surgical handling during subsequent implantation, but
which can then degrade rapidly in vivo. An alternative strategy would be to remove or uncrosslink the hydrogel once a sufficient amount of cartilaginous matrix has been deposited in vitro. Future studies in our lab will focus on various strategies to alter the degradation kinetics of the alginate material to accelerate endochondral bone formation, for instance by using gamma-irradiated, lower molecular weight alginate or partially oxidized alginate as this is known to increase the rate of degradation [24, 39, 42, 44]. Other approaches could include the incorporation of a more rapidly degrading second network within the alginate construct, the inclusion of a larger network of channels, the addition of inflammatory cytokines to enhance resorption, the chemical removal of the alginate phase entirely prior to implantation, or a reduction in the gelation time [36, 41, 45, 46]. As noted above, care needs to be taken to strike the correct balance between improving the degradation kinetics whilst ensuring the construct can remain intact during the in vitro culture phase and during surgical implantation.
4. Conclusions Scaling up of tissue engineering strategies to treat large bone defects remains a significant challenge in the field of orthopedic regenerative medicine. This study established the capability of alginate hydrogel material to support the in vitro chondrogenesis of MSCs and the subsequent initiation of neo-bone formation within large bone defects. Taken together, these results illustrate the capacity of chondrogenically primed MSC-alginate hydrogels to induce a similar healing response within critically-sized orthotopic bone defects of distinct embryological developmental pathways - the femur and the cranium. In both cases the chondrogenically primed MSC-alginate constructs appeared to actively support the deposition of new de novo bone appositionally along the surface of the engineered construct, and within regions of the implant that had degraded. Future work will focus on optimizing the
degradation kinetics of the hydrogel itself to accelerate de novo bone formation and facilitate complete regeneration of such defects.
Acknowledgements: This work was funded by the AO foundation under the large bone defect healing program. Surgical advice, PEEK fixator plates and custom designed surgical instruments were kindly provided by Professor Steve Goldstein, University of Michigan. Human bone marrow-derived MSCs were kindly provided by REMEDI, National Centre for Biomedical Engineering Science, National University of Ireland Galway, Galway, Ireland and funded by Science Foundation Ireland (grant number 09/SRC/B1794).
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Graphical abstract
Highlights: Alginate supports chondrogenesis of Mesenchymal Stem Cells Higher levels of mineralization observed in defects treated with engineered tissues Bone formation occurs appositional to the slowly degrading alginate hydrogel Hydrogel degradation rate regulates de novo bone deposition