Dana L. Nettles

Photocrosslinkable hyaluronan as a scaffold for articular cartilage repair.

Hyaluronan-based scaffolds are of interest for tissue-engineered cartilage repair due to an important role for hyaluronan in cartilage development and function. In this study, an in situ photocrosslinkable hyaluronan (HA-MA) was developed and evaluated as a scaffold for articular cartilage repair. Chondrocytes were encapsulated in crosslinked HA-MA and evaluated for their ability to synthesize cartilaginous matrix in vitro. The mechanical and physical properties of the crosslinked HA-MA hydrogels were similar to that of other hydrogels, with compressive and dynamic shear moduli of 0.6 and 0.3 kPa, respectively, and diffusion coefficients of 600–8000 μm2/s depending on molecular weight. Chondrocytes remained rounded in the HA-MA hydrogels in vitro, and accumulated significant amounts of cartilaginous matrix. Osteochondral defects filled with HA-MA were infiltrated with cells, appeared to integrate well with native tissue, and also accumulated substantial cartilaginous matrix by 2 weeks after surgery. In summary, photocrosslinkable HA-MA promoted the retention of the chondrocytic phenotype and cartilage matrix synthesis for encapsulated chondrocytes in vitro and accelerated healing in an in vivo osteochondral defect model.

Applications of elastin-like polypeptides in tissue engineering ☆

Elastin-like polypeptides (ELPs) have found utility in tissue engineering applications, not only because they are biocompatible, biodegradable, and non-immunogenic, but also because their amino acid sequence and molecular weight can be precisely controlled at the genetic or synthetic level, affording exquisite control over final protein functionality. This review presents a basic overview of ELP properties and modifications that are relevant to tissue engineering, as well as a discussion of the application of ELPs to cartilage, intervertebral disc, vascular graft, liver, ocular, and cell sheet engineering.

In situ cross-linking of elastin-like polypeptide block copolymers for tissue repair.

Rapid cross-linking of elastin-like polypeptides (ELPs) with hydroxymethylphosphines (HMPs) in aqueous solution is attractive for minimally invasive in vivo implantation of biomaterials and tissue engineering scaffolds. In order to examine the independent effect of the location and number of reactive sites on the chemical cross-linking kinetics of ELPs and the mechanical properties of the resulting hydrogels, we have designed ELP block copolymers comprised of cross-linkable, hydrophobic ELP blocks with periodic Lys residues (A block) and aliphatic, hydrophilic ELP blocks with no cross-linking sites (B block); three different block architectures, A, ABA, and BABA were synthesized in this study. All ELP block copolymers were rapidly cross-linked with HMPs within several minutes under physiological conditions. The inclusion of the un-cross-linked hydrophilic block, its length relative to the cross-linkable hydrophobic block, and the block copolymer architecture all had a significant effect on swelling ratios of the cross-linked hydrogels, their microstructure, and mechanical properties. Fibroblasts embedded in the ELP hydrogels survived the cross-linking process and remained viable for at least 3 days in vitro when the gels were formed from an equimolar ratio of HMPs and Lys residues of ELPs. DNA quantification of the embedded cells indicated that the cell viability within triblock ELP hydrogels was statistically greater than that in the monoblock gels at day 3. These results suggest that the mechanical properties of ELP hydrogels and the microenvironment that they present to cells can be tuned by the design of the block copolymer architecture.

Integrin expression in cells of the intervertebral disc

In this study, we investigated the profile of integrin expression in human and porcine intervertebral disc tissue. Differences in extracellular matrix composition between anulus fibrosus (AF) and nucleus pulposus (NP) regions of the disc, as well as differences in cellular responses to environmental stimuli, suggest a role for integrins in presenting matrix signals that may mediate these responses. Human disc tissue and porcine AF and NP tissue were stained with antibodies to alpha integrin subunits 1–6, V and IIb, and beta integrin subunits 1–6 and graded for evidence of positive staining on a scale from 0 (no staining) to 3 (high incidence of staining). Human tissue expressed α and β integrin subunits shown to be present in articular cartilage, including α1, α5 and αV. Porcine AF tissue expressed similar integrin subunits to human disc, with both expressing α1, α5, β1, β3 and β5 subunits, whereas porcine NP tissue expressed higher levels of α6, β1 and β4 than AF tissue. The expressed subunits are known to interact with proteins including collagens, fibronectin and laminin; however, additional studies will be required to characterize the interactions of the integrin subunits with specific matrix constituents, as well as their specific involvement in regulating environmental stimuli.

Sustained Release of Antibiotics from Injectable and Thermally Responsive Polypeptide Depots

Biodegradable polymeric scaffolds are of interest for delivering antibiotics to local sites of infection in orthopaedic applications, such as bone and diarthrodial joints. The objective of this study was to develop a biodegradable scaffold with ease of drug loading in aqueous solution, while providing for drug depot delivery via syringe injection. Elastin-like polypeptides (ELPs) were used for this application, biopolymers of repeating pentapeptide sequences that were thermally triggered to undergo in situ depot formation at body temperature. ELPs were modified to enable loading with the antibiotics, cefazolin, and vancomycin, followed by induction of the phase transition in vitro. Cefazolin and vancomycin concentrations were monitored, as well as bioactivity of the released antibiotics, to test an ability of the ELP depot to provide for prolonged release of bioactive drugs. Further tests of formulation viscosity were conducted to test suitability as an injectable drug carrier. Results demonstrate sustained release of therapeutic concentrations of bioactive antibiotics by the ELP, with first-order time constants for drug release of approximately 25 h for cefazolin and approximately 500 h for vancomycin. These findings illustrate that an injectable, in situ forming ELP depot can provide for sustained release of antibiotics with an effect that varies across antibiotic formulation. ELPs have important advantages for drug delivery, as they are known to be biocompatible, biodegradable, and elicit no known immune response. These benefits suggest distinct advantages over currently used carriers for antibiotic drug delivery in orthopedic applications.

Neural Network Analysis Identifies Scaffold Properties Necessary for In Vitro Chondrogenesis in Elastin-like Polypeptide Biopolymer Scaffolds

The successful design of biomaterial scaffolds for articular cartilage tissue engineering requires an understanding of the impact of combinations of material formulation parameters on diverse and competing functional outcomes of biomaterial performance. This study sought to explore the use of a type of unsupervised artificial network, a self-organizing map, to identify relationships between scaffold formulation parameters (crosslink density, molecular weight, and concentration) and 11 such outcomes (including mechanical properties, matrix accumulation, metabolite usage and production, and histological appearance) for scaffolds formed from crosslinked elastin-like polypeptide (ELP) hydrogels. The artificial neural network recognized patterns in functional outcomes and provided a set of relationships between ELP formulation parameters and measured outcomes. Mapping resulted in the best mean separation amongst neurons for mechanical properties and pointed to crosslink density as the strongest predictor of most outcomes, followed by ELP concentration. The map also grouped formulations together that simultaneously resulted in the highest values for matrix production, greatest changes in metabolite consumption or production, and highest histological scores, indicating that the network was able to recognize patterns amongst diverse measurement outcomes. These results demonstrated the utility of artificial neural network tools for recognizing relationships in systems with competing parameters, toward the goal of optimizing and accelerating the design of biomaterial scaffolds for articular cartilage tissue engineering.

Inflammatory Cytokines and Matrix Metalloproteinases in the Synovial Fluid After Intra-articular Ankle Fracture

Background: Posttraumatic osteoarthritis (PTOA) can occur after intra-articular fracture despite anatomic fracture reduction. It has been hypothesized that an early inflammatory response after intra-articular injury could lead to irreversible cartilage damage that progresses to PTOA. Therefore, in addition to meticulous fracture reduction, it would be ideal to prevent this initial inflammatory response but little is known about the composition of the synovial environment after intra-articular fracture. The purpose of this work was to characterize the inflammatory cytokine and matrix metalloproteinase (MMP) composition in the synovial fluid (SF) of patients with acute intra-articular ankle fractures. Methods: Twenty-one patients with an intra-articular ankle fracture were included in this study. All patients had a contralateral ankle joint that was pain free, had no radiographic evidence of arthritis, and no history of trauma. The uninjured ankle served as a matched control. SF was obtained from bilateral ankles at the time of surgery which occurred at a mean of 17 days post-fracture (range 8-40). The SF was analyzed for granulocyte macrophage colony-stimulating factor (GM-CSF), interferon-gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), interleukin (IL)-1β, IL-2, IL-6, IL-8, IL-10, IL-12p70, MMP-1, MMP-2, MMP-3, MMP-9, MMP-10, CTXII, sGAG, and bilirubin/biliverdin (markers of hemearthrosis) using either multiplex assay or ELISA using commercially available kits. Mean concentrations of each factor were compared between SF from fractured and control ankles, and correlation analysis was done to determine potential relationships between levels of cytokines and time from fracture and age at fracture. Results: Twelve of 18 measured factors including GM-CSF, IL-10, IL-1β, IL-6, IL-8, TNF-α, MMP-1, MMP-2, MMP-3, MMP-9, MMP-10, and bilirubin/biliverdin were found to be significantly higher in the fractured ankles. Mean concentrations of ECM degradation markers (sGAG and CTXII) were not found to be significatnly different between groups. Conclusion: These data indicate that after intra-articular ankle fracture the SF exhibits a largely pro-inflammatory and extra-cellular matrix degrading environment similar to that described in idiopathic osteoarthritis. IL-6, IL-8, MMP-1, MMP-2, MMP-3, MMP-9, and MMP-10 were significantly elevated and may play a role in the development of PTOA. Clinical Relevance: In addition to anatomic fracture reduction, these data lend credence to reducing acute intra-articular inflammation through the development of antagonists to these pro-inflammatory and degrading mediators. Likewise, intra-articular lavage might reduce this inflammatory burden.

Gait and behavior in an IL1β‐mediated model of rat knee arthritis and effects of an IL1 antagonist

Interleukin‐1 beta (IL1β) is a proinflammatory cytokine that mediates arthritic pathologies. Our objectives were to evaluate pain and limb dysfunction resulting from IL1β over‐expression in the rat knee and to investigate the ability of local IL1 receptor antagonist (IL1Ra) delivery to reverse‐associated pathology. IL1β over‐expression was induced in the right knees of 30 Wistar rats via intra‐articular injection of rat fibroblasts retrovirally infected with human IL1β cDNA. A subset of animals received a 30 µl intra‐articular injection of saline or human IL1Ra on day 1 after cell delivery (0.65 µg/µl hIL1Ra, n = 7 per group). Joint swelling, gait, and sensitivity were investigated over 1 week. On day 8, animals were sacrificed and joints were collected for histological evaluation. Joint inflammation and elevated levels of endogenous IL1β were observed in knees receiving IL1β‐infected fibroblasts. Asymmetric gaits favoring the affected limb and heightened mechanical sensitivity (allodynia) reflected a unilateral pathology. Histopathology revealed cartilage loss on the femoral groove and condyle of affected joints. Intra‐articular IL1Ra injection failed to restore gait and sensitivity to preoperative levels and did not reduce cartilage degeneration observed in histopathology. Joint swelling and degeneration subsequent to IL1β over‐expression is associated limb hypersensitivity and gait compensation. Intra‐articular IL1Ra delivery did not result in marked improvement for this model; this may be driven by rapid clearance of administered IL1Ra from the joint space. These results motivate work to further investigate the behavioral consequences of monoarticular arthritis and sustained release drug delivery strategies for the joint space.

Release and activity of anti-TNFα therapeutics from injectable chitosan preparations for local drug delivery

BACKGROUND Tumor necrosis factor alpha (TNFalpha) is a cytokine that regulates immune and inflammatory overactivation in various pathological states. Protein therapeutics may antagonize this cytokine, but may also have systemic toxicities. Small molecule natural products are also efficacious, but can suffer from poor oral bioavailability. A drug delivery vehicle is needed to sustain release of active therapeutics and address localized inflammation. MATERIALS Chitosan is a biocompatible aminopolysaccharide that undergoes thermally-initiated gelation in cosolutions with glycerophosphate (GP), and may entrap and sustain release of additive therapeutics. Gelation time and temperature of chitosan/GP were evaluated by turbidity (OD(350)), as was the kinetic effect of bovine serum albumin (BSA) entrapment. We investigated in vitro release of BSA and various anti-TNF agents (curcumin, sTNFRII, anti-TNF antibody) and confirmed in vitro activity of the released drugs using an established bioassay. RESULTS Turbidity results show that chitosan/GP thermogel achieves gelation at 37 degrees C within 10 min, even with significant protein loading. Sustained BSA release occurred with 50% retained at 7 days. All anti-TNF therapeutics exhibited sustained release, with 10% of sTNFRII and anti-TNF antibody remaining after 7 days and 10% of curcumin remaining after 20 days. After release, each compound antagonized TNFalpha-cytotoxicity in murine fibrosarcoma cells. CONCLUSIONS This study demonstrates that thermogelling chitosan/GP entraps and sustains release of a broad range of anti-TNF agents. Such delivery of disease-modifying therapy could establish a drug depot to treat local inflammation. The breadth of molecular sizes demonstrates significant versatility, and slow release could protect against toxicities of systemic delivery.

The role of metabolomics in osteoarthritis research.

Metabolomics is the comprehensive analysis of small molecules in a biological system and has generated great interest for identifying novel biomarkers for disease diagnosis and pharmaceutical treatment. Metabolomics has been used to identify citrate and choline as biomarkers for prostate and breast cancer, respectively. In fact, both tests are now clinically available and supported by most health insurance providers.1, 2 Metabolites are the end-products of cellular processes, and their levels can be regarded as the ultimate response of biological systems to genotype, phenotype, and environmental conditions. They encompass a diverse group of low-molecular weight compounds including lipids, amino acids, peptides, nucleic acids, organic acids, vitamins, thiols, and carbohydrates,3 and are commonly analyzed using nuclear magnetic resonance spectroscopy, liquid chromatography/mass spectrometry, and/or gas chromatography/mass spectrometry. Metabolomics may be well suited for osteoarthritis (OA) research for many reasons including the tremendous heterogeneity in the disease process and recognition that no single biomarker can reflect the breadth of temporal and pathological processes associated with OA.4 Combining several biomarkers into a panel will assuredly increase the discriminatory capability5, as would occur with metabolic profiling. Secondly, since metabolic perturbations are real-time, they indicate the current disease state, a distinct advantage over current clinical diagnostics and disease monitoring techniques for OA, such as radiography. Metabolomics has been employed to detect metabolic perturbations in urine, blood, synovium, and synovial fluid (SF) of animal models and patients with OA. Lamers and co-workers6 used nuclear magnetic resonance to study the urine from guinea pigs that spontaneously develop OA. Disturbances in lactic acid, malic acid, hypoxanthine and alanine were found to contribute heavily to the metabolic profile of OA. These investigators further studied the urine of humans with and without OA and found distinct patterns in the NMR spectra that could discriminate between groups.4 Zhai and co-workers7 employed metabolomics on human serum in a study of patients with and without knee OA. The investigators demonstrated that the ratios of valine and leucine to histidine were predictive of OA, pointing towards interest in the use of branched-chain amino acids (BCAA) as potential biomarkers.7 Many of these studies of venous plasma or urine identify metabolites that may be related to aging, altered muscle mass, and other factors that may confound the unique signature of a pathological OA joint. For these reasons, SF may yield the most accurate, real-time, and joint-specific metabolic profile. Metabolomics has been performed on SF from experimentally induced OA in canine knee joints.8 The nuclear magnetic resonance spectra demonstrated increased concentrations of lactate, pyruvate, glycerol, alanine, isoleucine, hydroxybutyrate, hydroxyisobutyrate, and lipoprotein associated fatty acids. The investigators concluded that the intra-articular environment of OA was more hypoxic and acidotic than in the normal joint, and that arthritic joints may rely in part upon altered lipid metabolic pathways. We have performed metabolomics on both human synovium and SF. In our first study, the metabolic profile of conditioned media collected from synovium explant cultures was obtained for tissues from patients undergoing total knee arthroplasty (end-stage OA) and from patients undergoing ligament or meniscal repair with little or no evidence of OA.9 Thirteen metabolites were significantly elevated in the end-stage OA group and included glutamine, succinate, pro-hydroxyproline, amongst others. Despite results suggestive of a distinct metabolic profile in OA, the synovium culture method does not easily translate into clinical practice. In a second study, we performed metabolomics on ankle SF of patients with and without ankle OA.10 Results identified 106 metabolites as significantly elevated in the OA samples and represented perturbations in virtually all metabolic pathways, including amino acid metabolism, carbohydrate metabolism, mitochondrial oxidation, lipid metabolism, peptide, vitamin, nucleotide synthesis, and redox homeostasis. More importantly, when a rigorous decision tree analysis was applied to the metabolic profiles of the two populations, a 90% discriminatory accuracy was achieved, indicating the potential use of this technology as a diagnostic tool for OA. Studies are ongoing to confirm these findings in a larger population and to generate a narrowed panel of metabolic biomarkers and measurement methods for translation into the clinic. In conclusion, metabolic profiling of biofluids and tissues can provide a panoramic view of the current physiologic state of a biological system such as the intra-articular environment of an osteoarthritic joint. We envision the role of metabolomics for OA as a clinically applied diagnostic tool in which a sample of a patient’s synovial fluid would be analyzed for a panel of metabolite biomarkers, similar to following serial values from a complete blood count. Alterations in the metabolic profile could indicate disease progression or a therapeutic response at a resolution not possible with currently employed clinical techniques.