Lower crosslinking density enhances functional nucleus pulposus-like matrix elaboration by human mesenchymal stem cells in carboxymethylcellulose hydrogels.

Engineered constructs represent a promising treatment for replacement of nucleus pulposus (NP) tissue. Recently, photocrosslinked hydrogels comprised of methacrylated carboxymethylcellulose (CMC) were shown to support chondrogenic differentiation of encapsulated human mesenchymal stem cells (hMSCs) and promote accumulation of NP-like extracellular matrix (ECM). The objective of this study was to investigate the influence of CMC crosslinking density, by varying macromer concentration and modification (i.e., methacrylation) percentage, on NP-like differentiation of encapsulated hMSCs. Constructs of lower macromer concentration (2%, w/v) exhibited significantly greater collagen II accumulation, more homogeneous distribution of ECM macromolecules, and a temporal increase in mechanical properties compared to hydrogels of higher macromer concentration (4%, w/v). Constructs of higher modification percentage (25%) gave rise to significantly elevated collagen II content and the formation of cell clusters within the matrix relative to samples of lower modification percentage (10% and 15%). These differences in functional ECM accumulation and distribution are likely attributed to the distinct crosslinked network structures of the various hydrogel formulations. Overall, CMC constructs of lower macromer concentration and modification percentage were most promising as scaffolds for NP tissue engineering based on functional ECM assembly. Optimization of such hydrogel fabrication parameters may lead to the development of clinically relevant tissue-engineered NP replacements.

Duration of TGF-ß3 Exposure Impacts the Chondrogenic Maturation of Human MSCs in Photocrosslinked Carboxymethylcellulose Hydrogels.

Intervertebral disc (IVD) herniation can be caused by both degeneration and traumatic injury, ultimately resulting in back pain or sciatica due to disc protrusion. Replacement of the nucleus pulposus (NP) tissue during surgical intervention post herniation could improve the long-term stability of the functional spinal unit. Tissue engineering strategies may potentially restore both biological and mechanical function of the NP. Recently, photocrosslinked carboxymethylcellulose (CMC) hydrogels were shown to support chondrogenic, NP-like extracellular matrix (ECM) elaboration by human mesenchymal stromal cells (hMSCs) when supplemented with TGF-ß3. However, long-term preconditioning with soluble growth factors in vitro or the use of sustained growth factor delivery vehicles in vivo can be expensive and difficult to control. Transient supplementation with growth factors has been shown to maintain or improve maturation of tissue-engineered constructs. The objective of this study was to evaluate the influence of TGF-ß3 exposure time on hydrogel bulk properties and NP-like matrix elaboration in hMSC-laden CMC hydrogels. Constructs were exposed to TGF-ß3 for 2 weeks (Transient), 8 weeks (Continuous) or 0 weeks (controls). After 8 weeks of culture, both the Transient and Continuous groups exhibited increased ECM accumulation compared to 2 weeks and controls. The Transient group displayed significantly greater accumulation of collagens I and II, while GAG content was significantly higher in the Continuous group by 8 weeks. Distribution of ECM was more homogeneous in the Continuous group, while the Transient group exhibited more concentrated accumulation in the periphery of the hydrogel by 8 weeks. Mechanical properties improved over time in both groups, however, Continuous constructs demonstrated significantly more robust mechanical properties (equilibrium modulus and peak stress) compared to Transient gels at 8 weeks. Although the functional properties of Transient constructs did not surpass those achieved by Continuous scaffolds, they increased and were maintained upon growth factor removal at 2 weeks, and were greater than controls. Additionally, Transient construct mechanical properties (equilibrium modulus, % relaxation) were similar to those of native NP tissue. The differences seen in ECM distribution and subsequent construct functional maturation are likely due to the time available for diffusion of growth factors through the construct. Overall, these findings support the use of short-term TGF-ß3 treatment to promote sufficient long-term tissue maturation in vitro in this hMSC-laden CMC hydrogel system.

Functional nucleus pulposus-like matrix assembly by human mesenchymal stromal cells is directed by macromer concentration in photocrosslinked carboxymethylcellulose hydrogels.

Intervertebral disc (IVD) degeneration is associated with several pathophysiologic changes of the IVD, including dehydration of the nucleus pulposus (NP). Tissue engineering strategies may be used to restore both biological and mechanical function of the IVD following removal of NP tissue during surgical intervention. Recently, photocrosslinked carboxymethylcellulose (CMC) hydrogels were shown to support chondrogenic, NP-like extracellular matrix (ECM) elaboration by human mesenchymal stromal cells (hMSCs) when supplemented with TGF-ß3; however, mechanical properties of these constructs did not reach native values. Fabrication parameters (i.e., composition, crosslinking density) can influence the bulk mechanical properties of hydrogel scaffolds, as well as cellular behavior and differentiation patterns. The objective of this study was to evaluate the influence of CMC macromer concentration (1.5, 2.5 and 3.5 % weight/volume) on bulk hydrogel properties and NP-like matrix elaboration by hMSCs. The lowest macromer concentration of 1.5 % exhibited the highest gene expression levels of aggrecan and collagen II at day 7, corresponding with the largest accumulation of glycosaminoglycans and collagen II by day 42. The ECM elaboration in the 1.5 % constructs was more homogeneously distributed compared to primarily pericellular localization in 3.5 % gels. The 1.5 % gels also displayed significant improvements in mechanical functionality by day 42 compared to earlier time points, which was not seen in the other groups. The effects of macromer concentration on matrix accumulation and organization are likely attributed to quantifiable differences in polymer crosslinking density and diffusive properties between the various hydrogel formulations. Taken together, these results demonstrate that macromer concentration of CMC hydrogels can direct hMSC matrix elaboration, such that a lower polymer concentration allows for greater NP-like ECM assembly and improvement of mechanical properties over time.

Injectable redox-polymerized methylcellulose hydrogels as potential soft tissue filler materials.

There is a significant clinical need for long-lasting, injectable materials for soft tissue reconstruction. Methylcellulose (MC) is an FDA-approved polysaccharide derivative of cellulose that is inexpensive, renewable, and biocompatible, and may serve as an alternative to existing synthetic and natural fillers. In this study, MC was modified with functional methacrylate groups and polymerized using a redox-initiation system to produce hydrogels with tunable properties. By varying the percent methacrylation and macromer concentration, the equilibrium moduli of the hydrogels were found to range between 1.29 ± 0.46 and 12.8 ± 2.94 kPa, on par with human adipose tissue, and also displayed an inverse relationship to the swelling properties. Rheological analyses determined gelation onset and completion to be in accordance with the ISO standard for injectable materials. Cellulase enzymatic treatment resulted in complete degradation of the hydrogels by 48 h, presenting the possibility of minimally invasive removal of the materials in the event of malposition or host reaction. In addition, co-culture experiments with human dermal fibroblasts showed the gels to be cytocompatible based on DNA measurements and Live/Dead staining. Taken together, these redox-polymerized MC hydrogels may be of use for a wide range of clinical indications requiring soft tissue augmentation.

Role of biomechanics in intervertebral disc degeneration and regenerative therapies: what needs repairing in the disc and what are promising biomaterials for its repair?

BACKGROUND CONTEXT: Degeneration and injuries of the intervertebral disc (IVD) result in large alterations in biomechanical behaviors. Repair strategies using biomaterials can be optimized based on the biomechanical and biological requirements of the IVD. PURPOSE: To review the present literature on the effects of degeneration, simulated degeneration, and injury on biomechanics of the IVD, with special attention paid to needle puncture injuries, which are a pathway for diagnostics and regenerative therapies and the promising biomaterials for disc repair with a focus on how those biomaterials may promote biomechanical repair. STUDY DESIGN: A narrative review to evaluate the role of biomechanics on disc degeneration and regenerative therapies with a focus on what biomechanical properties need to be repaired and how to evaluate and accomplish such repairs using biomaterials. Model systems for the screening of such repair strategies are also briefly described. METHODS: Articles were selected from two main PubMed searches using keywords: intervertebral AND biomechanics (1,823 articles) and intervertebral AND biomaterials (361 articles). Additional keywords (injury, needle puncture, nucleus pressurization, biomaterials, hydrogel, sealant, tissue engineering) were used to narrow the articles down to the topics most relevant to this review. RESULTS: Degeneration and acute disc injuries have the capacity to influence nucleus pulposus (NP) pressurization and annulus fibrosus (AF) integrity, which are necessary for an effective disc function and, therefore, require repair. Needle injection injuries are of particular clinical relevance with the potential to influence disc biomechanics, cellularity, and metabolism, yet these effects are localized or small and more research is required to evaluate and reduce the potential clinical morbidity using such techniques. NP replacement strategies, such as hydrogels, are required to restore the NP pressurization or the lost volume. AF repair strategies including cross-linked hydrogels, fibrous composites, and sealants offer promise for regenerative therapies to restore AF integrity. Tissue engineered IVD structures, as a single implantable construct, may promote greater tissue integration due to the improved repair capacity of the vertebral bone. CONCLUSIONS: IVD height, neutral zone characteristics, and torsional biomechanics are sensitive to specific alterations in the NP pressurization and AF integrity and must be addressed for an effective functional repair. Synthetic and natural biomaterials offer promise for NP replacement, AF repair, as an AF sealant, or whole disc replacement. Meeting mechanical and biological compatibilities are necessary for the efficacy and longevity of the repair.

Transforming growth factor-beta 3 stimulates cartilage matrix elaboration by human marrow-derived stromal cells encapsulated in photocrosslinked carboxymethylcellulose hydrogels: potential for nucleus pulposus replacement.

Degeneration of the nucleus pulposus (NP) has been implicated as a major cause of low back pain. Tissue engineering strategies using marrow-derived stromal cells (MSCs) have been used to develop cartilaginous tissue constructs, which may serve as viable NP replacements. Supplementation with growth factors, such as transforming growth factor-beta 3 (TGF-ß3), has been shown to enhance the differentiation of MSCs and promote functional tissue development of such constructs. A potential candidate material that may be useful as a scaffold for NP tissue engineering is carboxymethylcellulose (CMC), a biocompatible, cost-effective derivative of cellulose. Photocrosslinked CMC hydrogels have been shown to support NP cell viability and promote phenotypic matrix deposition capable of maintaining mechanical properties when cultured in serum-free, chemically defined medium (CDM) supplemented with TGF-ß3. However, MSCs have not been characterized using this hydrogel system. In this study, human MSCs (hMSCs) were encapsulated in photocrosslinked CMC hydrogels and cultured in CDM with and without TGF-ß3 to determine the effect of the growth factor on the differentiation of hMSCs toward an NP-like phenotype. Constructs were evaluated for matrix elaboration and functional properties consistent with native NP tissue. CDM supplemented with TGF-ß3 resulted in significantly higher glycosaminoglycan content (762.69±220.79 ng/mg wet weight) and type II collagen (COL II) content (6.25±1.64 ng/mg wet weight) at day 21 compared with untreated samples. Immunohistochemical analyses revealed uniform, pericellular, and interterritorial staining for chondroitin sulfate proteoglycan and COL II in growth factor-supplemented constructs compared with faint, strictly pericellular staining in untreated constructs at 21 days. Consistent with matrix deposition, mechanical properties of hydrogels treated with TGF-ß3 increased over time and exhibited the highest peak stress in stress-relaxation (σ(pk)=1.489±0.389 kPa) at day 21 among all groups. Taken together, these results demonstrate that hMSCs encapsulated in photocrosslinked CMC hydrogels supplemented with TGF-ß3 are capable of elaborating functional extracellular matrix consistent with the NP phenotype. Such MSC-laden hydrogels may have application in NP replacement therapies.