Histological and Inflammatory Cytokine Analysis of Osteochondral Lesions of the Talus After Failed Microfracture: Comparison With Fresh Allograft Controls.

BACKGROUND: The most common first-line treatment of osteochondral lesions of the talus (OLTs) is microfracture. Although many patients do well with this procedure, a number fail and require reoperation. The mechanism of failure of microfracture is unknown, and to our knowledge there has been no research characterizing failed microfracture regarding histological and inflammatory makeup of these lesions that may contribute to failure. PURPOSE: To characterize the structural and biochemical makeup of failed microfracture lesions. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: Specimens from 8 consecutive patients with symptomatic OLTs after microfracture who later underwent fresh osteochondral allograft transplantation were analyzed. For each patient, the failed microfracture specimen and a portion of the fresh allograft replacement tissue were collected. The allograft served as a control. Histology of the failed microfracture and the allograft replacement was scored using the Osteoarthritis Research Society International (OARSI) system. Surface roughness was also compared. In addition, tissue culture supernatants were analyzed for 16 secreted cytokines and matrix metalloproteinases (MMPs) responsible for inflammation, pain, cartilage damage, and chondrocyte death. RESULTS: The OARSI grade, stage, and total score as well as surface smoothness were significantly worse in the failed microfracture sample, indicating better cartilage and bone morphology for the allografts compared with the failed microfracture lesions. Analyzed cytokines and MMPs were significantly elevated in the microfracture tissue culture supernatants when compared with fresh osteochondral tissue supernatants. CONCLUSION: These data demonstrate a significantly rougher cartilage surface, cartilage and subchondral bone histology that more closely resembles osteoarthritis, and elevated inflammatory cytokines and MMPs responsible for pain, inflammation, cartilage damage, and chondrocyte death when compared with fresh osteochondral allografts used as controls.

Inflammatory cytokines and matrix metalloproteinases in the synovial fluid after intra-articular elbow fracture.

BACKGROUND AND HYPOTHESIS: Post-traumatic elbow contracture remains a common and challenging complication with often unsatisfactory outcomes. Although the etiology is unknown, elevated or abnormal post-fracture synovial fluid cytokine levels may result in the migration of fibroblasts to the capsule and contribute to capsular pathology. Thus, the purpose of this study was to characterize the cytokine composition in the synovial fluid fracture hematoma of patients with intra-articular elbow fractures. METHODS: The elbow synovial fluid fracture hematoma of 11 patients with intra-articular elbow fractures was analyzed for CTXII (C-terminal telopeptides of type II collagen [a cartilage breakdown product]) as well as 15 cytokines and matrix metalloproteinases (MMPs) including interferon γ, interleukin (IL) 1ß, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, tumor necrosis factor α, MMP-1, MMP-2, MMP-3, MMP-9, and MMP-10. The uninjured, contralateral elbow served as a matched control. Mean concentrations of each factor were compared between the fluid from fractured elbows and the fluid from control elbows. RESULTS: The levels of 14 of 15 measured cytokines and MMPs-interferon γ, IL-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, tumor necrosis factor α, MMP-1, MMP-3, MMP-9, and MMP-10-were significantly higher in the fractured elbows. In addition, post hoc power analysis revealed that 10 of 14 significant differences were detected with greater than 90% power. The mean concentration of CTXII was not significantly different between groups. CONCLUSIONS: These results demonstrate a proinflammatory environment after fracture that may be the catalyst to the development of post-traumatic elbow joint contracture. The cytokines with elevated levels were similar, although not identical, to the cytokines with elevated levels in studies of other weight-bearing joints, indicating the elbow responds uniquely to trauma.

Five-Year Follow-Up of Distal Tibia Bone and Foot and Ankle Trauma Treated with a 3D-Printed Titanium Cage.

Large bone defects from trauma or cancer are difficult to treat. Current treatment options include the use of external fixation with bone transport, bone grafting, or amputation. These modes of therapy continue to pose challenges as they are associated with high cost, failure, and complication rates. In this study, we report a successful case of bone defect treatment using personalized 3D-printed implant. This is the longest known follow-up using a 3D-printed custom implant for this specific application. Ultimately, this report adds to existing literature as it demonstrates successful and maintained incorporation of bone into the titanium implant. The use of patient-specific 3D-printed implants adds to the available arsenal to treat complex pathologies of the foot and ankle. Moreover, the technology's flexibility and ease of customization makes it conducive to tailor to specific patient needs.

Amino Acid Profile of Synovial Fluid Following Intra-articular Ankle Fracture.

BACKGROUND: Post-traumatic osteoarthritis (PTOA) is a frequent complication in patients with a previous traumatic joint injury, and the pathophysiology is not well understood. The goal of this study was to characterize the biochemical signature of amino acids, peptides, and amino acid metabolites in ankle synovial fluid following intra-articular fracture. METHODS: Synovial fluid from both the injured and contralateral ankles of 19 patients with an intra-articular ankle fracture was obtained and analyzed via metabolic profiling. Follow-up analysis was performed after 6 months in 7 of these patients. RESULTS: Statistical comparisons between injured and contralateral ankles revealed that 19 of the 66 measured amino acids, peptides, and amino acid metabolites were significantly elevated at time of fracture. Metabolites associated with glutathione metabolism exhibited the most elevated mean-fold changes, indicating a possible role for oxidative stress in fractured ankles. None of the metabolites elevated at baseline were significantly elevated after 6 months, but 6 metabolites had mean-fold changes greater than 2.1 at this time point. Multiple metabolites also exhibited significant correlations ( r > 0.575) with matrix metalloproteinase-1 and -9. CONCLUSION: These results indicate the presence of amino acid metabolic products in the setting of ankle fracture and suggest that these changes in amino acid metabolism may be chronic and indicate a role for inflammation and collagen degradation in disease progression. CLINICAL RELEVANCE: Changes in amino acid metabolism following intra-articular fracture may contribute to the progression to PTOA. This knowledge may allow for the identification and early treatment of patients at risk of developing PTOA. LEVEL OF EVIDENCE: Level III, comparative series.

Time-Dependent Effects on Synovial Fluid Composition During the Acute Phase of Human Intra-articular Ankle Fracture.

BACKGROUND: The study objective was to examine the effect of time and fracture severity on the undiluted synovial fluid (SF) microenvironment during the acute phase following intra-articular fracture (IAF) of the human ankle. METHODS: Ankle SF from 54 patients with an acute IAF was analyzed for concentrations of 10 cytokines, 5 matrix metalloproteinases, 2 products of cartilage catabolism, and combined products of heme metabolism. All analytes were correlated with time from fracture and further analyzed for an effect of 3 time subgroups (0-2 days, 3-9 days, and ≥10 days) corresponding to timepoints for clinical ankle fracture interventions. The effect of fracture severity was determined by grouping SF according to the number of radiographic intra-articular fracture lines. RESULTS: Fifteen of 18 analytes were significantly correlated with time. Temporal grouping of SF revealed an initial (0-2 days) spike of pro-inflammatory (IL-12p70, IL-1ß, IL-6) and anti-inflammatory (IL-10 and IL-4) cytokines, matrix metalloproteinases (MMP) MMP-9, and sGAG, followed immediately (3-9 days) by products of heme metabolism and an unchallenged surge in mediators and products of cartilage catabolism (MMP-1, MMP-2, MMP-3, MMP-10, and CTX-II). After 10 days, there was a decrease in pro- and anti-inflammatory cytokines but a persistence of mediators of ECM catabolism. There was no clear relationship between the number of fracture lines and SF levels of analytes. CONCLUSIONS: This study demonstrated acute temporal fluctuations following ankle IAF resulting in an overall catabolic environment by 10 days post-fracture and supports consideration of an early evacuation of the joint space to reduce the intra-articular inflammatory burden. Clinical Relavence: This study contributes to the understanding of the intra-articular events that potentially contribute to the development of posttraumatic osteoarthritis acutely following IAF in the ankle.

Inflammatory Microenvironment Persists After Bone Healing in Intra-articular Ankle Fractures.

BACKGROUND: Post-traumatic osteoarthritis (PTOA) is responsible for the majority of cases of ankle arthritis. While acute and end-stage intra-articular inflammation has previously been described, the state of the joint between fracture healing and end-stage PTOA remains undefined. This study characterized synovial fluid (SF) composition of ankles after bone healing of an intra-articular fracture to identify factors that may contribute to the development of PTOA. METHODS: Of an original 21 patients whose SF was characterized acutely following intra-articular ankle fractures, 7 returned for planned hardware (syndesmotic screw) removal after bone healing (approximately 6 months) and consented to a second bilateral SF collection. SF concentrations of 15 cytokines and matrix metalloproteinases (MMPs) and 2 markers each of cartilage catabolism (CTXII and glycosaminoglycan) and hemarthrosis (biliverdin and bilirubin) were compared for previously fractured and contralateral, uninjured ankles from the same patient. Analysis was also performed to determine the effect of the number of fracture lines and involvement of soft tissue on SF composition. RESULTS: Interleukin (IL)-6, IL-8, MMP-1, MMP-2, and MMP-3 were significantly elevated in the SF from healed ankles compared to matched contralateral uninjured ankles at approximately 6 months after fracture. There were no differences in markers of cartilage catabolism or hemarthrosis. Only IL-1α was affected by the number of fracture lines while differences were not detected for other analytes or with respect to the involvment of soft tissue. CONCLUSIONS: Sustained intra-articular inflammation, even after complete bone healing, was suggested by elevations of pro-inflammatory cytokines (IL-6 and IL-8). In addition, elevated concentrations of MMPs were also noted and were consistent with a persistent inflammatory environment. This study suggests new evidence of persistent intra-articular inflammation after intra-articular ankle fracture healing and suggests potential mediators for PTOA development. CLINICAL RELEVANCE: This work may be relevant to the clinical diagnosis and treatment of post-traumatic osteoarthritis.

Lipid profile of human synovial fluid following intra-articular ankle fracture.

This study characterizes the metabolic profile of synovial fluid after intra-articular ankle fracture with an emphasis on changes in the lipid profile. Bilateral ankle synovial fluid from 19 patients with unilateral intra-articular ankle fracture was submitted for metabolic profiling. Contralateral ankle synovial fluid from each patient served as a matched control. Seven patients participated in a second bilateral synovial fluid collection after 6 months. Random forest classification, matched pairs t-tests (α < 0.01), repeated measures ANOVA with post-test contrasts (α < 0.01), correlation to cytokines and matrix metalloproteinases, and fracture and injury classification analyses yielded key lipid biomarkers in synovial fluid following intra-articular fracture. Free fatty acids, sphingomyelins, and lysolipids demonstrated significant elevation in fractured ankles at baseline. Fatty acids and sphingomyelins showed a significant decrease 6 months post-surgery. Random forest analysis showed predominantly fatty acids differentiating between groups. Significant correlations included fatty acids, sphingomyelins, and lysolipids with inflammatory cytokines and matrix metalloproteinases. Fracture classification showed increased fatty acids, lysolipids, and inositol metabolites as fracture severity increased. Fatty acid and sn-1 lysolipid elevation could be detrimental to the joint, as these strongly correlated with matrix metalloproteinases and TNF-α. This elevation also suggests involvement of phospholipase A2 , a potential target for therapeutic intervention. Together with elevated 2-hydroxyl fatty acids, these findings suggest elevated sn-1 lysolipids, sphingomyelins, and subsequent lipid metabolites in synovial fluid as biomarkers of ankle injury. Reversal of this signature after 6 months suggests temporary involvement of these metabolites in disease progression, although they may activate signaling pathways which drive progression to osteoarthritis. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:657-666, 2017.

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)-1ß, 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-1ß, 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.

Inflammatory cytokines and cellular metabolites as synovial fluid biomarkers of posttraumatic ankle arthritis.

BACKGROUND: There is a paucity of research on posttraumatic ankle arthritis (PTAA). We aimed to identify synovial fluid PTAA biomarkers using cytokine analysis and metabolic profiling. METHODS: Ankle joint synovial fluid was obtained from 20 patients with PTAA and 20 patients with no ankle pain and no radiographic evidence of ankle arthritis (control group). Synovial fluid samples were analyzed for IFN-γ, TNF-α, MIP-1ß, MCP-1, IL-1ß, IL-1Ra, IL-4, IL-6, IL-8, IL-10, IL-13, and IL-15 using ELISA and for more than 3000 metabolites using liquid and gas chromatography with mass spectroscopy. To compare presence of cytokines and metabolites between groups, t tests were used. Random forest analysis was performed on metabolites to determine whether control and PTAA samples could be differentiated based on metabolic profile. RESULTS: IL-1Ra, IL-6, IL-8, IL-10, IL-15, and MCP-1 were significantly elevated in the PTAA group. In addition, 107 metabolites in the PTAA group were significantly altered, including derangement in amino acid, carbohydrate, lipid, and energy metabolism, extracellular matrix turnover, and collagen degradation. Random forest analysis yielded a predictive accuracy of 90% when using the metabolic profiles to distinguish between control and PTAA samples. CONCLUSION: This study identified inflammatory cytokines and metabolites present in the synovial fluid of PTAA. CLINICAL RELEVANCE: Several of these entities have previously been implicated in rheumatoid arthritis and osteoarthritis of the knee, but many could potentially be used as novel biomarkers of PTAA. Most importantly, the findings suggest that metabolites could be used to distinguish synovial fluid from patients with PTAA.

Gait and behavior in an IL1ß-mediated model of rat knee arthritis and effects of an IL1 antagonist.

Interleukin-1 beta (IL1ß) is a proinflammatory cytokine that mediates arthritic pathologies. Our objectives were to evaluate pain and limb dysfunction resulting from IL1ß over-expression in the rat knee and to investigate the ability of local IL1 receptor antagonist (IL1Ra) delivery to reverse-associated pathology. IL1ß over-expression was induced in the right knees of 30 Wistar rats via intra-articular injection of rat fibroblasts retrovirally infected with human IL1ß 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 IL1ß were observed in knees receiving IL1ß-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 IL1ß 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.

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.

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.

Early metabolite levels predict long-term matrix accumulation for chondrocytes in elastin-like polypeptide biopolymer scaffolds.

The development of cartilage tissue engineering scaffolds could greatly benefit from methods to evaluate the interactions of cells with scaffolds that are rapid, are nondestructive, and can be carried out at early culture times. Motivated by this rationale, the objective of the current study was to evaluate whether the concentration of metabolites in scaffold-cell cultures at early culture times could predict matrix synthesis in the same samples at longer culture times. Metabolite and matrix synthesis were measured for 16 different formulations of cell-laden elastin-like polypeptide hydrogels. Metabolites were measured at days 4 and 7 of culture, while matrix accumulation was evaluated at day 28. Four of the 16 formulations resulted in molar ratios of lactate:glucose near 2, indicating anaerobic metabolism of glucose, which resulted in collagen:glycosaminoglycan accumulation ratios near those of native tissue. Lactate and pyruvate concentrations were found to significantly correlate with both sulfated glycosaminoglycan and hydroxyproline accumulation, with better fits for the latter. Lactate was found to be the strongest predictor of both matrix components, suggesting that measuring this metabolite at very early culture times may be useful for evaluating the status of tissue engineering constructs in a rapid and nondestructive manner.

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.

Release and activity of anti-TNFalpha 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.

In situ crosslinking elastin-like polypeptide gels for application to articular cartilage repair in a goat osteochondral defect model.

The objective of this study was to evaluate an injectable, in situ crosslinkable elastin-like polypeptide (ELP) gel for application to cartilage matrix repair in critically sized defects in goat knees. One cylindrical, osteochondral defect in each of seven animals was filled with an aqueous solution of ELP and a biocompatible, chemical crosslinker, while the contralateral defect remained unfilled and served as an internal control. Joints were sacrificed at 3 (n = 3) or 6 (n = 4) months for MRI, histological, and gross evaluation of features of biomaterial performance, including integration, cellular infiltration, surrounding matrix quality, and new matrix in the defect. At 3 months, ELP-filled defects scored significantly higher for integration by histological and gross grading compared to unfilled defects. ELP did not impede cell infiltration but appeared to be partly degraded. At 6 months, new matrix in unfilled defects outpaced that in ELP-filled defects and scored significantly better for MRI evidence of adverse changes, as well as integration and proteoglycan-containing matrix via gross and histological grading. The ELP-crosslinker solution was easily delivered and formed stable, well-integrated gels that supported cell infiltration and matrix synthesis; however, rapid degradation suggests that ELP formulation modifications should be optimized for longer-term benefits in cartilage repair applications.

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.

Rapid cross-linking of elastin-like polypeptides with (hydroxymethyl)phosphines in aqueous solution.

In situ gelation of injectable polypeptide-based materials is attractive for minimally invasive in vivo implantation of biomaterials and tissue engineering scaffolds. We demonstrate that chemically cross-linked elastin-like polypeptide (ELP) hydrogels can be rapidly formed in aqueous solution by reacting lysine-containing ELPs with an organophosphorous cross-linker, beta-[tris(hydroxymethyl)phosphino]propionic acid (THPP) under physiological conditions. The mechanical properties of the cross-linked ELP hydrogels were largely modulated by the molar concentration of lysine residues in the ELP and the pH at which the cross-linking reaction was carried out. Fibroblasts embedded in ELP hydrogels survived the cross-linking process and were viable after in vitro culture for 3 days. DNA quantification of ELP hydrogels with encapsulated fibroblasts indicated that there was no significant difference in DNA content between day 0 and day 3 when ELP hydrogels were formed with an equimolar ratio of THPP and lysine residues of the ELPs. These results suggest that THPP cross-linking may be a biocompatible strategy for the in situ formation of cross-linked hydrogels.

Biodendrimer-based hydrogel scaffolds for cartilage tissue repair.

Photo-crosslinkable dendritic macromolecules are attractive materials for the preparation of cartilage tissue engineering scaffolds that may be optimized for in situ formation of hydrated, mechanically stable, and well-integrated hydrogel scaffolds supporting chondrocytes and chondrogenesis. We designed and synthesized a novel hydrogel scaffold for cartilage repair, based on a multivalent and water-soluble tri-block copolymer consisting of a poly(ethylene glycol) core and methacrylated poly(glycerol succinic acid) dendrimer terminal blocks. The terminal methacrylates allow mild and biocompatible photo-crosslinking with a visible light, facilitating in vivo filling of irregularly shaped defects with the dendrimer-based scaffold. The multivalent dendrimer constituents allow high crosslink densities that inhibit swelling after crosslinking while simultaneously introducing biodegradation sites. The mechanical properties and water content of the hydrogel can easily be tuned by changing the biodendrimer concentration. In vitro chondrocyte encapsulation studies demonstrate significant synthesis of neocartilaginous material, containing proteoglycans and type II collagen.