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.

Combining adhesive and nonadhesive injectable hydrogels for intervertebral disc repair in an ovine discectomy model.

Background: Intervertebral disc (IVD) disorders (e.g., herniation) directly contribute to back pain, which is a leading cause of global disability. Next-generation treatments for IVD herniation need advanced preclinical testing to evaluate their ability to repair large defects, prevent reherniation, and limit progressive degeneration. This study tested whether experimental, injectable, and nonbioactive biomaterials could slow IVD degeneration in an ovine discectomy model. Methods: Ten skeletally mature sheep (4-5.5 years) experienced partial discectomy injury with cruciate-style annulus fibrosus (AF) defects and 0.1 g nucleus pulposus (NP) removal in the L1-L2, L2-L3, and L3-L4 lumbar IVDs. L4-L5 IVDs were Intact controls. IVD injury levels received: (1) no treatment (Injury), (2) poly (ethylene glycol) diacrylate (PEGDA), (3) genipin-crosslinked fibrin (FibGen), (4) carboxymethylcellulose-methylcellulose (C-MC), or (5) C-MC and FibGen (FibGen + C-MC). Animals healed for 12 weeks, then IVDs were assessed using computed tomography (CT), magnetic resonance (MR) imaging, and histopathology. Results: All repaired IVDs retained ~90% of their preoperative disc height and showed minor degenerative changes by Pfirrmann grading. All repairs had similar disc height loss and Pfirrmann grade as Injury IVDs. Adhesive AF sealants (i.e., PEGDA and FibGen) did not herniate, although repair caused local endplate (EP) changes and inflammation. NP repair biomaterials (i.e., C-MC) and combination repair (i.e., FibGen + C-MC) exhibited lower levels of degeneration, less EP damage, and less severe inflammation; however, C-MC showed signs of herniation via biomaterial expulsion. Conclusions: All repair IVDs were noninferior to Injury IVDs by IVD height loss and Pfirrmann grade. C-MC and FibGen + C-MC IVDs had the best outcomes, and may be appropriate for enhancement with bioactive factors (e.g., cells, growth factors, and miRNAs). Such bioactive factors appear to be necessary to prevent injury-induced IVD degeneration. Application of AF sealants alone (i.e., PEGDA and FibGen) resulted in EP damage and inflammation, particularly for PEGDA IVDs, suggesting further material refinements are needed.

Differential surface antigen expression and 1α,25-dihydroxyvitamin D3 responsiveness distinguish human dermal fibroblasts with age-dependent osteogenic differentiation potential from marrow-derived stromal cells in vitro.

BACKGROUND AIMS: Recent studies have demonstrated that cells committed to a fibroblastic lineage, including dermal fibroblasts, may undergo osteoblastic differentiation when treated with steroid hormones. However, stem cells have also been isolated from the dermis, making it unclear whether osteoinduction of dermal fibroblasts is the result of transdifferentiation of committed fibroblasts or differentiation of resident multipotent stromal cells, which are morphologically indistinguishable. METHODS: Flow cytometry was used to characterize the expression of CD26, CD90 and CD105 on neonatal and adult human dermal fibroblasts and adult human bone marrow-derived stromal cells. These cells were then cultured with the steroid hormones 1α,25-dihydroxyvitamin D(3) and dexamethasone, and evaluated for protein expression and mineral deposition typical of an osteoblastic phenotype. RESULTS: The surface peptidase, dipeptidyl peptidase IV (CD26), was differentially expressed between human neonatal (98.22 ± 1.47%) and adult (90.73 ± 7.97%) dermal fibroblasts and adult bone marrow-derived stromal cells (6.84 ± 5.07%). In addition, neonatal dermal fibroblasts treated with vitamin D(3) expressed alkaline phosphatase, osteocalcin and bone sialoprotein, and deposited mineral, which is consistent with an osteoblastic phenotype. Such differentiation was not observed in adult dermal fibroblasts. In contrast, marrow-derived stromal cells required dexamethasone in order to undergo osteoblastic differentiation. CONCLUSIONS: Taken together, the differential surface antigen expression and disparate response to steroid hormones suggest that committed neonatal dermal fibroblasts are distinct from mesenchymal stromal cells and possess osteogenic differentiation potential.

Serum-free, chemically defined medium with TGF-beta(3) enhances functional properties of nucleus pulposus cell-laden carboxymethylcellulose hydrogel constructs.

Degeneration of the nucleus pulposus (NP) has been implicated as a major cause of low back pain. Tissue engineering strategies may provide a viable NP replacement therapy; however, culture conditions must be optimized to promote functional tissue development. In this study, a standard serum-containing medium formulation was compared to a chemically defined, serum-free medium to determine the effect on matrix elaboration and functional properties of NP cell-laden carboxymethylcellulose (CMC) hydrogels. Additionally, both media were further supplemented with transforming growth factor-beta 3 (TGF-beta(3)). Glycosaminoglycan (GAG) content increased in both TGF-beta(3)-treated groups and was highest for treated, serum-free constructs (9.46 +/- 1.51 microg GAG/mg wet weight), while there were no quantifiable GAGs in untreated serum-containing samples. Histology revealed uniform, interterritorial staining for chondroitin sulfate proteoglycan throughout the treated, serum-free constructs. Type II collagen content was greater in both serum-free groups and highest in treated, serum-free constructs. The equilibrium Young's modulus was highest in serum-free samples supplemented with TGF-beta(3) (18.54 +/- 1.92 kPa), and the equilibrium weight swelling ratio of these constructs approached that of the native NP tissue (22.19 +/- 0.46 vs. 19.94 +/- 3.09, respectively). Taken together, these results demonstrate enhanced functional matrix development by NP cells when cultured in CMC hydrogels maintained in serum-free, TGF-beta(3) supplemented medium, indicating the importance of medium formulation in NP construct development.

A rat model of temporomandibular joint pain with histopathologic modifications.

AIMS: To develop a rat model of temporomandibular joint (TMJ) pain and to characterize in it the development and temporal response of behavioral hypersensitivity as well as to evaluate if and to what extent a loading protocol is associated with histological changes in the TMJ consistent with osteoarthritic pathology. METHODS: A novel rat model of TMJ pain was developed using a noninvasive, mechanical loading protocol. Rats were exposed to steady mouth-opening for 7 days (2 N force, 1 hour/day), and mechanical hyperalgesia (increased pain response) was measured during the loading period and for 14 days thereafter. Histological modifications in the joint cartilage were also evaluated. Outcomes for the mouth-opening exposure were compared to age-matched controls. Thresholds for evoking responses were compared using a ranked ANOVA with repeated measures. RESULTS: Increased mechanical hypersensitivity in the temporomandibular region developed during daily loading and persisted even after the termination of the loading protocol. Histologic characterization revealed thinning of the cartilaginous structures of the joint and irregular zonal cellular arrangements in the condylar cartilage of rats subjected to the daily loading protocol. CONCLUSION: The injury model presented here is the first to demonstrate mechanically-induced behavioral hypersensitivity accompanied by osteoarthritic pathology in the TMJ.

Development of a two-part biomaterial adhesive strategy for annulus fibrosus repair and ex vivo evaluation of implant herniation risk.

Intervertebral disc (IVD) herniation causes pain and disability, but current discectomy procedures alleviate pain without repairing annulus fibrosus (AF) defects. Tissue engineering strategies seal AF defects by utilizing hydrogel systems to prevent recurrent herniation, however current biomaterials are limited by poor adhesion to wetted tissue surfaces or low failure strength resulting in considerable risk of implant herniation upon spinal loading. Here, we developed a two-part repair strategy comprising a dual-modified (oxidized and methacrylated) glycosaminoglycan that can chemically adsorb an injectable interpenetrating network hydrogel composed of fibronectin-conjugated fibrin and poly (ethylene glycol) diacrylate (PEGDA) to covalently bond the hydrogel to AF tissue. We show that dual-modified hyaluronic acid imparts greater adhesion to AF tissue than dual-modified chondroitin sulfate, where the degree of oxidation is more strongly correlated with adhesion strength than methacrylation. We apply this strategy to an ex vivo bovine model of discectomy and demonstrate that PEGDA molecular weight tunes hydrogel mechanical properties and affects herniation risk, where IVDs repaired with low-modulus hydrogels composed of 20kDa PEGDA failed at levels at or exceeding discectomy, the clinical standard of care. This strategy bonds injectable hydrogels to IVD extracellular matrix proteins, is optimized to seal AF defects, and shows promise for IVD repair.

Injectable cellulose-based hydrogels as nucleus pulposus replacements: Assessment of in vitro structural stability, ex vivo herniation risk, and in vivo biocompatibility.

Current treatments for intervertebral disc degeneration and herniation are palliative only and cannot restore disc structure and function. Nucleus pulposus (NP) replacements are a promising strategy for restoring disc biomechanics and height loss. Cellulose-based hydrogel systems offer potential for NP replacement since they are stable, non-toxic, may be tuned to match NP material properties, and are conducive to cell or drug delivery. A crosslinked, carboxymethylcellulose-methylcellulose dual-polymer hydrogel was recently formulated as an injectable NP replacement that gelled in situ and restored disc height and compressive biomechanical properties. The objective of this study was to investigate the translational potential of this hydrogel system by examining the long-term structural stability in vitro, the herniation risk and fatigue bending endurance in a bovine motion segment model, and the in vivo biocompatibility in a rat subcutaneous pouch model. Results showed that the hydrogels maintained their structural integrity over a 12-week period. AF injury significantly increased herniation risk and reduced fatigue bending endurance in bovine motion segments. Samples repaired with cellulosic hydrogels demonstrated restored height and exhibited herniation risk and fatigue endurance comparable to samples that underwent the current standard treatment of nucleotomy. Lastly, injected hydrogels elicited a minimal foreign body response as determined by analysis of fibrous capsule development and macrophage presence over 12 weeks. Overall, this injectable cellulosic hydrogel system is a promising candidate as an NP substitute. Further assessment and optimization of this cellulosic hydrogel system in an in vivo intradiscal injury model may lead to an improved clinical solution for disc degeneration and herniation.

Composite biomaterial repair strategy to restore biomechanical function and reduce herniation risk in an ex vivo large animal model of intervertebral disc herniation with varying injury severity.

Back pain commonly arises from intervertebral disc (IVD) damage including annulus fibrosus (AF) defects and nucleus pulposus (NP) loss. Poor IVD healing motivates developing tissue engineering repair strategies. This study evaluated a composite injectable IVD biomaterial repair strategy using carboxymethylcellulose-methylcellulose (CMC-MC) and genipin-crosslinked fibrin (FibGen) that mimic NP and AF properties, respectively. Bovine ex vivo caudal IVDs were evaluated in cyclic compression-tension, torsion, and compression-to-failure tests to determine IVD biomechanical properties, height loss, and herniation risk following experimentally-induced severe herniation injury and discectomy (4 mm biopsy defect with 20% NP removed). FibGen with and without CMC-MC had failure strength similar to discectomy injury suggesting no increased risk compared to surgical procedures, yet no biomaterials improved axial or torsional biomechanical properties suggesting they were incapable of adequately restoring AF tension. FibGen had the largest failure strength and was further evaluated in additional discectomy injury models with varying AF defect types (2 mm biopsy, 4 mm cruciate, 4 mm biopsy) and NP removal volume (0%, 20%). All simulated discectomy defects significantly compromised failure strength and biomechanical properties. The 0% NP removal group had mean values of axial biomechanical properties closer to intact levels than defects with 20% NP removed but they were not statistically different and 0% NP removal also decreased failure strength. FibGen with and without CMC-MC failed at super-physiological stress levels above simulated discectomy suggesting repair with these tissue engineered biomaterials may perform better than discectomy alone, although restored biomechanical function may require additional healing with the potential application of these biomaterials as sealants and cell/drug delivery carriers.

Fetal ACL fibroblasts exhibit enhanced cellular properties compared with adults.

Fetal tendons and skin heal regeneratively without scar formation. Cells isolated from these fetal tissues exhibit enhanced cellular migration and collagen production in comparison to cells from adult tissue. We determined whether fetal and adult fibroblasts isolated from the anterior cruciate ligament (ACL), a tissue that does not heal regeneratively, exhibit differences in cell migration rates and collagen elaboration. An in vitro migration assay showed fetal ACL fibroblasts migrated twice as fast as adult ACL fibroblasts at a rate of 38.90 +/- 7.69 microm per hour compared with 18.88 +/- 4.18 microm per hour, respectively. Quantification of Type I collagen elaboration by enzyme-linked immunosorbent assay showed fetal ACL fibroblasts produced four times the amount of Type I collagen compared with adult ACL fibroblasts after 7 days in culture. We observed no differences in Type III collagen with time for adult or fetal ACL fibroblasts. Our findings indicate fetal ACL fibroblasts are intrinsically different from adult ACL fibroblasts, suggesting the healing potential of the ACL may be age-dependent.

Downregulation of Heat Shock Protein 70 Impairs Osteogenic and Chondrogenic Differentiation in Human Mesenchymal Stem Cells.

Human mesenchymal stem cells (hMSCs) show promise for bone and cartilage regeneration. Our previous studies demonstrated that hMSCs with periodic mild heating had enhanced osteogenic and chondrogenic differentiation with significantly upregulated heat shock protein 70 (HSP70). However, the role of HSP70 in adult tissue regeneration is not well studied. Here, we revealed an essential regulatory mechanism of HSP70 in osteogenesis and chondrogenesis using adult hMSCs stably transfected with specific shRNAs to knockdown HSP70. Periodic heating at 39 °C was applied to hMSCs for up to 26 days. HSP70 knockdown resulted in significant reductions of alkaline phosphatase activity, calcium deposition, and gene expression of Runx2 and Osterix during osteogenesis. In addition, knockdown of HSP70 led to significant decreases of collagens II and X during chondrogenesis. Thus, downregulation of HSP70 impaired hMSC osteogenic and chondrogenic differentiation as well as the enhancement of these processes by thermal treatment. Taken together, these findings suggest a putative mechanism of thermal-enhanced bone and cartilage formation and underscore the importance of HSP70 in adult bone and cartilage differentiation.

Injectable, redox-polymerized carboxymethylcellulose hydrogels promote nucleus pulposus-like extracellular matrix elaboration by human MSCs in a cell density-dependent manner.

Low back pain is a major cause for disability and is closely linked to intervertebral disc degeneration. Mechanical and biological dysfunction of the nucleus pulposus in the disc has been found to initiate intradiscal degenerative processes. Replacing or enriching the diseased nucleus pulposus with an injectable, stem cell-laden biomaterial that mimics its material properties can provide a minimally invasive strategy for biological and structural repair of the tissue. In this study, injectable, in situ-gelling carboxymethylcellulose hydrogels were developed for nucleus pulposus tissue engineering using encapsulated human marrow-derived mesenchymal stromal cells (hMSCs). With the goal of obtaining robust extracellular matrix deposition and faster construct maturation, two cell-seeding densities, 20 × 106 cells/ml and 40 × 106 cells/ml, were examined. The constructs were fabricated using a redox initiation system to yield covalently crosslinked, cell-seeded hydrogels via radical polymerization. Chondrogenic culture of the hydrogels over 35 days exhibited high cell viability along with deposition of proteoglycan and collagen-rich extracellular matrix, and mechanical and swelling properties similar to native human nucleus pulposus. Further, the matrix production and distribution in the carboxymethylcellulose hydrogels was found to be strongly influenced by hMSC-seeding density, with the lower cell-seeding density yielding a more favorable nucleus pulposus-specific matrix phenotype, while the rate of construct maturation was less dependent on the cell-seeding density. These findings are the first to demonstrate the utility of redox-polymerized carboxymethylcellulose hydrogels as hMSC carriers for potential minimally invasive treatment strategies for nucleus pulposus replacement.

Influence of three-dimensional scaffold on the expression of osteogenic differentiation markers by human dermal fibroblasts.

Current research in the field of tissue engineering utilizes biomaterial scaffolds, cells, and growth factors for the creation of a functional, biologically active tissue. This study examined the effect of two commercially available, three-dimensional scaffolds, ultraporous beta-tricalcium phosphate ceramics (beta-TCP, Vitoss) and open-celled poly(lactic acid) foams (OPLA, Drilac), on the osteogenic differentiation potential of human dermal fibroblasts. Serum-free, chemically-defined medium containing the metabolic factor 1alpha,25-dihydroxyvitamin D3 was used to promote an osteogenic phenotype in these cells. Osteoblast differentiation was assessed using PCR and immunohistochemical methods to detect gene and protein expression for the osteoblast markers alkaline phosphatase, osteopontin, and osteocalcin. Dermal fibroblasts cultured on beta-TCP scaffolds in chemically-defined medium with vitamin D3 exhibited up-regulated gene and protein expression compared to cells cultured on OPLA scaffolds. These results suggest that Vitoss (beta-TCP) scaffolds seeded with dermal fibroblasts and maintained in chemically-defined medium with vitamin D3 are better suited for bone tissue engineering applications than Drilac (OPLA) foams.

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.

Influence of serum on adult and fetal dermal fibroblast migration, adhesion, and collagen expression.

The wound healing response to injury can be affected by many factors such as cell migration and extracellular matrix elaboration. The objective of this study was to examine the serum- and age-dependent effects on cell migration, adhesion, and collagen expression by skin fibroblasts. Dermal fibroblasts were isolated and plated with and without serum for up to 7 d. Cell migration was determined by quantitative image analysis, adhesion was quantified using a centrifugation assay, and collagen expression was assessed by PCR and immunohistochemical staining. Both adult and fetal fibroblasts migrated significantly faster in serum-containing medium compared to serum-free medium. There was no significant difference in migration between the two cell types in either serum-containing or serum-free medium. There was no significant difference in adhesion in the presence of serum, although there was a greater fraction of adherent fetal skin fibroblasts than adult fibroblasts in serum-free medium. Moreover, the adherent fraction of fetal fibroblasts in serum-free medium was not significantly different from that in serum-containing medium, suggesting that fetal skin fibroblasts possess serum-independent adhesion properties. Collagen mRNA expression was significantly up-regulated in serum-free compared to serum-containing medium for both cell types. With respect to collagen immunohistochemistry, both dermal fibroblast populations exhibited greater type I collagen compared to type III collagen staining. Quantitative assessment of collagen staining indicated significantly enhanced type I collagen secretion in the presence of serum by fetal skin fibroblasts. These findings suggest that intrinsic cellular characteristics may govern the observed differences in adult and fetal wound healing.

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.

Development of crosslinked methylcellulose hydrogels for soft tissue augmentation using an ammonium persulfate-ascorbic acid redox system.

Hydrogels composed of methylcellulose are candidate materials for soft tissue reconstruction. Although photocrosslinked methylcellulose hydrogels have shown promise for such applications, gels crosslinked using reduction-oxidation (redox) initiators may be more clinically viable. In this study, methylcellulose modified with functional methacrylate groups was polymerized using an ammonium persulfate (APS)-ascorbic acid (AA) redox initiation system to produce injectable hydrogels with tunable properties. By varying macromer concentration from 2% to 4% (w/v), the equilibrium moduli of the hydrogels ranged from 1.47 ± 0.33 to 5.31 ± 0.71 kPa, on par with human adipose tissue. Gelation time was found to conform to the ISO standard for injectable materials. Cellulase treatment resulted in complete degradation of the hydrogels within 24h, providing a reversible corrective feature. Co-culture with human dermal fibroblasts confirmed the cytocompatibility of the gels based on DNA measurements and Live/Dead imaging. Taken together, this evidence indicates that APS-AA redox-polymerized methylcellulose hydrogels possess properties beneficial for use as soft tissue fillers.

Synergistic action of growth factors and dynamic loading for articular cartilage tissue engineering.

It has previously been demonstrated that dynamic deformational loading of chondrocyte-seeded agarose hydrogels over the course of 1 month can increase construct mechanical and biochemical properties relative to free-swelling controls. The present study examines the manner in which two mediators of matrix biosynthesis, the growth factors TGF-beta1 and IGF-I, interact with applied dynamic deformational loading. Under free-swelling conditions in control medium (C), the [proteoglycan content][collagen content][equilibrium aggregate modulus] of cell-laden (10 x 10(6) cells/mL) 2% agarose constructs reached a peak of [0.54% wet weight (ww)][0.16% ww][13.4 kPa]c, whereas the addition of TGF-beta1 or IGF-I to the control medium led to significantly higher peaks of [1.18% ww][0.97% ww][23.6 kPa](C-TGF) and [1.00% ww][0.63% ww][19.3 kPa](C-IGF), respectively, by day 28 or 35 (p<0.01). Under dynamic loading in control medium (L), the measured parameters were [1.10% ww][0.52% ww][24.5 kPa]L, and with the addition of TGF-beta1 or IGF-I to the control medium these further increased to [1.49% ww][1.07% ww][50.5 kPa](L-TGF) and [1.48% ww][0.81% ww][46.2 kPa](L-IGF), respectively (p<0.05). Immunohistochemical staining revealed that type II collagen accumulated primarily in the pericellular area under free-swelling conditions, but spanned the entire tissue in dynamically loaded constructs. Applied in concert, dynamic deformational loading and TGF-beta1 or IGF-I increased the aggregate modulus of engineered constructs by 277 or 245%, respectively, an increase greater than the sum of either stimulus applied alone. These results support the hypothesis that the combination of chemical and mechanical promoters of matrix biosynthesis can optimize the growth of tissue-engineered cartilage constructs.

Cartilage interstitial fluid load support in unconfined compression.

Under physiological conditions of loading, articular cartilage is subjected to both compressive strains, normal to the articular surface, and tensile strains, tangential to the articular surface. Previous studies have shown that articular cartilage exhibits a much higher modulus in tension than in compression, and theoretical analyses have suggested that this tension-compression nonlinearity enhances the magnitude of interstitial fluid pressurization during loading in unconfined compression, above a theoretical threshold of 33% of the average applied stress. The first hypothesis of this experimental study is that the peak fluid load support in unconfined compression is significantly greater than the 33% theoretical limit predicted for porous permeable tissues modeled with equal moduli in tension and compression. The second hypothesis is that the peak fluid load support is higher at the articular surface side of the tissue samples than near the deep zone, because the disparity between the tensile and compressive moduli is greater at the surface zone. Ten human cartilage samples from six patellofemoral joints, and 10 bovine cartilage specimens from three calf patellofemoral joints were tested in unconfined compression. The peak fluid load support was measured at 79 +/- 11% and 69 +/- 15% at the articular surface and deep zone of human cartilage, respectively, and at 94 +/- 4% and 71 +/- 8% at the articular surface and deep zone of bovine calf cartilage, respectively. Statistical analyses confirmed both hypotheses of this study. These experimental results suggest that the tension-compression nonlinearity of cartilage is an essential functional property of the tissue which makes interstitial fluid pressurization the dominant mechanism of load support in articular cartilage.

Carboxymethylcellulose hydrogels support central nervous system-derived tumor-cell chemotactic migration: comparison with conventional extracellular matrix macromolecules.

The local microenvironment plays an important role in maintaining the dynamics of the extracellular matrix and the cell-extracellular matrix relationship. The extracellular matrix is a complex network of macromolecules with distinct mechanical and biochemical characteristics. Disruptions in extracellular matrix homeostasis are associated with the onset of cancer. The extracellular matrix becomes highly disorganized, and the cell-matrix relationship changes, resulting in altered cell-signaling processes and metastasis. Medulloblastoma is one of the most common malignant pediatric brain tumors in the United States. In order to gain a better understanding of the interplay between cell-extracellular matrix interactions and cell-migratory responses in tumors, eight different matrix macromolecule formulations were investigated using a medulloblastoma-derived cell line: poly-D-lysine, matrigel, laminin, collagen 1, fibronectin, a 10% blend of laminin-collagen 1, a 20% blend of laminin-collagen 1, and a cellulose-derived hydrogel, carboxymethylcellulose. Over time, the average changes in cell morphology were quantified in 2D and 3D, as was migration in the presence and absence of the chemoattractant, epidermal growth factor. Data revealed that carboxymethylcellulose allowed for a cell-extracellular matrix relationship typically believed to be present in tumors, with cells exhibiting a rounded, amoeboid morphology consistent with chemotactic migration, while the other matrices promoted an elongated cell shape as well as both haptotactic and chemotactic motile processes. Therefore, carboxymethylcellulose hydrogels may serve as effective platforms for investigating central nervous system-derived tumor-cell migration in response to soluble factors.

Injectable carboxymethylcellulose hydrogels for soft tissue filler applications.

Disease, trauma and aging all lead to deficits in soft tissue. As a result, there is a need to develop materials that safely and effectively restore areas of deficiency. While autogenous fat is the current gold standard, hyaluronic acid (HA) fillers are commonly used. However, the animal and bacterial origin of HA-based materials can induce adverse reactions in patients. With the aim of developing a safer and more affordable alternative, this study characterized the properties of a plant-derived, injectable carboxymethylcellulose (CMC) soft tissue filler. Specifically, methacrylated CMC was synthesized and crosslinked to form stable hydrogels at varying macromer concentrations (2-4% w/v) using an ammonium persulfate and ascorbic acid redox initiation system. The equilibrium Young's modulus was shown to vary with macromer concentration (ranging from ∼2 to 9.25kPa), comparable to values of native soft tissue and current surgical fillers. The swelling properties were similarly affected by macromer concentration, with 4% gels exhibiting the lowest swelling ratio and mesh size, and highest crosslinking density. Rheological analysis was performed to determine gelation onset and completion, and was measured to be within the ISO standard for injectable materials. In addition, hydrolytic degradation of these gels was sensitive to macromer concentration, while selective removal using enzymatic treatment was also demonstrated. Moreover, favorable cytocompatibility of the CMC hydrogels was exhibited by co-culture with human dermal fibroblasts. Taken together, these findings demonstrate the tunability of redox-crosslinked CMC hydrogels by varying fabrication parameters, making them a versatile platform for soft tissue filler applications.