A BMP/Activin A Chimera Induces Posterolateral Spine Fusion in Nonhuman Primates at Lower Concentrations Than BMP-2.

BACKGROUND: Supraphysiologic bone morphogenetic protein (BMP)-2 concentrations are required to induce spinal fusion. In this study, a BMP-2/BMP-6/activin A chimera (BV-265), optimized for BMP receptor binding, delivered in a recombinant human collagen:CDHA [calcium-deficient hydroxyapatite] porous composite matrix (CM) or bovine collagen:CDHA granule porous composite matrix (PCM), engineered for optimal BV-265 retention and guided tissue repair, was compared with BMP-2 delivered in a bovine absorbable collagen sponge (ACS) wrapped around a MASTERGRAFT Matrix (MM) ceramic-collagen rod (ACS:MM) in a nonhuman primate noninstrumented posterolateral fusion (PLF) model. METHODS: In vivo retention of 125I-labeled-BV-265/CM or PCM was compared with 125I-labeled-BMP-2/ACS or BMP-2/buffer in a rat muscle pouch model using scintigraphy. Noninstrumented PLF was performed by implanting CM, BV-265/CM, BV-265/PCM, or BMP-2/ACS:MM across L3-L4 and L5-L6 or L3-L4-L5 decorticated transverse processes in 26 monkeys. Computed tomography (CT) images were acquired at 0, 4, 8, 12, and 24 weeks after surgery, where applicable. Manual palpation, µCT (microcomputed tomography) or nCT (nanocomputed tomography), and histological analysis were performed following euthanasia. RESULTS: Retention of 125I-labeled-BV-265/CM was greater than BV-265/PCM, followed by BMP-2/ACS and BMP-2/buffer. The CM, 0.43 mg/cm3 BMP-2/ACS:MM, and 0.05 mg/cm3 BV-265/CM failed to generate PLFs. The 0.15-mg/cm3 BV-265/CM or 0.075-mg/cm3 BV-265/PCM combinations were partially effective. The 0.25-mg/cm3 BV-265/CM and 0.15 and 0.3-mg/cm3 BV-265/PCM combinations generated successful 2-level PLFs at 12 and 24 weeks. CONCLUSIONS: BV-265/CM or PCM can induce fusion in a challenging nonhuman primate noninstrumented PLF model at substantially lower concentrations than BMP-2/ACS:MM. CLINICAL RELEVANCE: BV-265/CM and PCM represent potential alternatives to induce PLF in humans at substantially lower concentrations than BMP-2/ACS:MM.

A BMP/activin A chimera is superior to native BMPs and induces bone repair in nonhuman primates when delivered in a composite matrix.

Bone morphogenetic protein (BMP)/carriers approved for orthopedic procedures achieve efficacy superior or equivalent to autograft bone. However, required supraphysiological BMP concentrations have been associated with potential local and systemic adverse events. Suboptimal BMP/receptor binding and rapid BMP release from approved carriers may contribute to these outcomes. To address these issues and improve efficacy, we engineered chimeras with increased receptor binding by substituting BMP-6 and activin A receptor binding domains into BMP-2 and optimized a carrier for chimera retention and tissue ingrowth. BV-265, a BMP-2/BMP-6/activin A chimera, demonstrated increased binding affinity to BMP receptors, including activin-like kinase-2 (ALK2) critical for bone formation in people. BV-265 increased BMP intracellular signaling, osteogenic activity, and expression of bone-related genes in murine and human cells to a greater extent than BMP-2 and was not inhibited by BMP antagonist noggin or gremlin. BV-265 induced larger ectopic bone nodules in rats compared to BMP-2 and was superior to BMP-2, BMP-2/6, and other chimeras in nonhuman primate bone repair models. A composite matrix (CM) containing calcium-deficient hydroxyapatite granules suspended in a macroporous, fenestrated, polymer mesh-reinforced recombinant human type I collagen matrix demonstrated improved BV-265 retention, minimal inflammation, and enhanced handling. BV-265/CM was efficacious in nonhuman primate bone repair models at concentrations ranging from 1/10 to 1/30 of the BMP-2/absorbable collagen sponge (ACS) concentration approved for clinical use. Initial toxicology studies were negative. These results support evaluations of BV-265/CM as an alternative to BMP-2/ACS in clinical trials for orthopedic conditions requiring augmented healing.

Sustained delivery of bioactive TGF-ß1 from self-assembling peptide hydrogels induces chondrogenesis of encapsulated bone marrow stromal cells.

Tissue engineering strategies for cartilage defect repair require technology for local targeted delivery of chondrogenic and anti-inflammatory factors. The objective of this study was to determine the release kinetics of transforming growth factor ß1 (TGF-ß1) from self-assembling peptide hydrogels, a candidate scaffold for cell transplant therapies, and stimulate chondrogenesis of encapsulated young equine bone marrow stromal cells (BMSCs). Although both peptide and agarose hydrogels retained TGF-ß1, fivefold higher retention was found in peptide. Excess unlabeled TGF-ß1 minimally displaced retained radiolabeled TGF-ß1, demonstrating biologically relevant loading capacity for peptide hydrogels. The initial release from acellular peptide hydrogels was nearly threefold lower than agarose hydrogels, at 18% of loaded TGF-ß1 through 3 days as compared to 48% for agarose. At day 21, cumulative release of TGF-ß1 was 32-44% from acellular peptide hydrogels, but was 62% from peptide hydrogels with encapsulated BMSCs, likely due to cell-mediated TGF-ß1 degradation and release of small labeled species. TGF-ß1 loaded peptide hydrogels stimulated chondrogenesis of young equine BMSCs, a relevant preclinical model for treating injuries in young human cohorts. Self-assembling peptide hydrogels can be used to deliver chondrogenic factors to encapsulated cells making them a promising technology for in vivo, cell-based regenerative medicine.

Regional variations in the distribution and colocalization of extracellular matrix proteins in the juvenile bovine meniscus.

A deeper understanding of the composition and organization of extracellular matrix molecules in native, healthy meniscus tissue is required to fully appreciate the degeneration that occurs in joint disease and the intricate environment in which an engineered meniscal graft would need to function. In this study, regional variations in the tissue-level and pericellular distributions of collagen types I, II and VI and the proteoglycans aggrecan, biglycan and decorin were examined in the juvenile bovine meniscus. The collagen networks were extensively, but not completely, colocalized, with tissue-level organization that varied with radial position across the meniscus. Type VI collagen exhibited close association with large bundles composed of type I and II collagen and, in contrast to type I and II collagen, was further concentrated in the pericellular matrix. Aggrecan was detected throughout the inner region of the meniscus but was restricted to the pericellular matrix and sheaths of collagen bundles in the middle and outer regions. The small proteoglycans biglycan and decorin exhibited regional variations in staining intensity but were consistently localized in the intra- and/or peri-cellular compartments. These results provide insight into the complex hierarchy of extracellular matrix organization in the meniscus and provide a framework for better understanding meniscal degeneration and disease progression and evaluating potential repair and regeneration strategies.

Controlled delivery of transforming growth factor ß1 by self-assembling peptide hydrogels induces chondrogenesis of bone marrow stromal cells and modulates Smad2/3 signaling.

Self-assembling peptide hydrogels were modified to deliver transforming growth factor ß1 (TGF-ß1) to encapsulated bone-marrow-derived stromal cells (BMSCs) for cartilage tissue engineering applications using two different approaches: (i) biotin-streptavidin tethering; (ii) adsorption to the peptide scaffold. Initial studies to determine the duration of TGF-ß1 medium supplementation necessary to stimulate chondrogenesis showed that 4 days of transient soluble TGF-ß1 to newborn bovine BMSCs resulted in 10-fold higher proteoglycan accumulation than TGF-ß1-free culture after 3 weeks. Subsequently, BMSC-seeded peptide hydrogels with either tethered TGF-ß1 (Teth-TGF) or adsorbed TGF-ß1 (Ads-TGF) were cultured in the TGF-ß1-free medium, and chondrogenesis was compared to that for BMSCs encapsulated in unmodified peptide hydrogels, both with and without soluble TGF-ß1 medium supplementation. Ads-TGF peptide hydrogels stimulated chondrogenesis of BMSCs as demonstrated by cell proliferation and cartilage-like extracellular matrix accumulation, whereas Teth-TGF did not stimulate chondrogenesis. In parallel experiments, TGF-ß1 adsorbed to agarose hydrogels stimulated comparable chondrogenesis. Full-length aggrecan was produced by BMSCs in response to Ads-TGF in both peptide and agarose hydrogels, whereas medium-delivered TGF-ß1 stimulated catabolic aggrecan cleavage product formation in agarose but not peptide scaffolds. Smad2/3 was transiently phosphorylated in response to Ads-TGF but not Teth-TGF, whereas medium-delivered TGF-ß1 produced sustained signaling, suggesting that dose and signal duration are potentially important for minimizing aggrecan cleavage product formation. Robustness of this technology for use in multiple species and ages was demonstrated by effective chondrogenic stimulation of adult equine BMSCs, an important translational model used before the initiation of human clinical studies.

Tensile loading modulates bone marrow stromal cell differentiation and the development of engineered fibrocartilage constructs.

Mesenchymal progenitors such as bone marrow stromal cells (BMSCs) are an attractive cell source for fibrocartilage tissue engineering, but the types or combinations of signals required to promote fibrochondrocyte-specific differentiation remain unclear. The present study investigated the influences of cyclic tensile loading on the chondrogenesis of BMSCs and the development of engineered fibrocartilage. Cyclic tensile displacements (10%, 1 Hz) were applied to BMSC-seeded fibrin constructs for short (24 h) or extended (1-2 weeks) periods using a custom loading system. At early stages of chondrogenesis, 24 h of cyclic tension stimulated both protein and proteoglycan synthesis, but at later stages, tension increased protein synthesis only. One week of intermittent cyclic tension significantly increased the total sulfated glycosaminoglycan and collagen contents in the constructs, but these differences were lost after 2 weeks of loading. Constraining the gels during the extended culture periods prevented contraction of the fibrin matrix, induced collagen fiber alignment, and increased sulfated glycosaminoglycan release to the media. Cyclic tension specifically stimulated collagen I mRNA expression and protein synthesis, but had no effect on collagen II, aggrecan, or osteocalcin mRNA levels. Overall, these studies suggest that the combination of chondrogenic stimuli and tensile loading promotes fibrochondrocyte-like differentiation of BMSCs and has the potential to direct fibrocartilage development in vitro.

Mechanical injury of explants from the articulating surface of the inner meniscus.

Knee osteoarthritis is accelerated by damage to the meniscus, a fibrocartilage tissue that assists in load transmission. However, little is known about the mechanical or cellular response of the meniscus to injurious overloading. Here, in vitro studies explored injury to meniscal explants using a compressive overloading protocol that has been well characterized for articular cartilage. Cartilage samples were processed in parallel as a reference to the extensive literature on cartilage injury. Injured meniscal explants showed extensive cell death at the articulating surface but no gross tissue damage, while similar conditions of peak stress and strain resulted in cartilage surface fissures and cell death consistent with moderate overloading. Post-injury gene expression in meniscal explants indicated a decrease in seven of the nine catabolic and pro-inflammatory molecules surveyed, while cartilage experienced a downregulation in ADAMTS-5 and TNF-alpha only. These data demonstrated a resiliency of the meniscus to injury, and that an acute increase in catabolic activities is not necessarily a consequence of mechanical overloading.

Self-assembling peptide hydrogels modulate in vitro chondrogenesis of bovine bone marrow stromal cells.

Our objective was to test the hypothesis that self-assembling peptide hydrogel scaffolds provide cues that enhance the chondrogenic differentiation of bone marrow stromal cells (BMSCs). BMSCs were encapsulated within two unique peptide hydrogel sequences, and chondrogenesis was compared with that in agarose hydrogels. BMSCs in all three hydrogels underwent transforming growth factor-beta1-mediated chondrogenesis as demonstrated by comparable gene expression and biosynthesis of extracellular matrix molecules. Expression of an osteogenic marker was unchanged, and an adipogenic marker was suppressed by transforming growth factor-beta1 in all hydrogels. Cell proliferation occurred only in the peptide hydrogels, not in agarose, resulting in higher glycosaminoglycan content and more spatially uniform proteoglycan and collagen type II deposition. The G1-positive aggrecan produced in peptide hydrogels was predominantly the full-length species, whereas that in agarose was predominantly the aggrecanase product G1-NITEGE. Unique cell morphologies were observed for BMSCs in each peptide hydrogel sequence, with extensive cell-cell contact present for both, whereas BMSCs in agarose remained rounded over 21 days in culture. Differences in cell morphology within the two peptide scaffolds may be related to sequence-specific cell adhesion. Taken together, this study demonstrates that self-assembling peptide hydrogels enhance chondrogenesis compared with agarose as shown by extracellular matrix production, DNA content, and aggrecan molecular structure.

Aggrecanolysis and in vitro matrix degradation in the immature bovine meniscus: mechanisms and functional implications.

INTRODUCTION: Little is known about endogenous or cytokine-stimulated aggrecan catabolism in the meniscal fibrocartilage of the knee. The objectives of this study were to characterize the structure, distribution, and processing of aggrecan in menisci from immature bovines, and to identify mechanisms of extracellular matrix degradation that lead to changes in the mechanical properties of meniscal fibrocartilage. METHODS: Aggrecanase activity in the native immature bovine meniscus was examined by immunolocalization of the aggrecan NITEGE neoepitope. To investigate mechanisms of cytokine-induced aggrecan catabolism in this tissue, explants were treated with interleukin-1alpha (IL-1) in the absence or presence of selective or broad spectrum metalloproteinase inhibitors. The sulfated glycosaminoglycan (sGAG) and collagen contents of explants and culture media were quantified by biochemical methods, and aggrecan catabolism was examined by Western analysis of aggrecan fragments. The mechanical properties of explants were determined by dynamic compression and shear tests. RESULTS: The aggrecanase-generated NITEGE neoepitope was preferentially localized in the middle and outer regions of freshly isolated immature bovine menisci, where sGAG density was lowest and blood vessels were present. In vitro treatment of explants with IL-1 triggered the accumulation of NITEGE in the inner and middle regions. Middle region explants stimulated with IL-1 exhibited substantial decreases in sGAG content, collagen content, and mechanical properties. A broad spectrum metalloproteinase inhibitor significantly reduced sGAG loss, abrogated collagen degradation, and preserved tissue mechanical properties. In contrast, an inhibitor selective for ADAMTS-4 and ADAMTS-5 was least effective at blocking IL-1-induced matrix catabolism and loss of mechanical properties. CONCLUSIONS: Aggrecanase-mediated aggrecanolysis, typical of degenerative articular cartilage, may play a physiologic role in the development of the immature bovine meniscus. IL-1-induced release of sGAG and loss of mechanical properties can be ascribed primarily to the activity of MMPs or aggrecanases other than ADAMTS-4 and ADAMTS-5. These results may have implications for the clinical management of osteoarthritis.

Mechanical injury potentiates proteoglycan catabolism induced by interleukin-6 with soluble interleukin-6 receptor and tumor necrosis factor alpha in immature bovine and adult human articular cartilage.

OBJECTIVE: Traumatic joint injury can damage cartilage and release inflammatory cytokines from adjacent joint tissue. The present study was undertaken to study the combined effects of compression injury, tumor necrosis factor alpha (TNFalpha), and interleukin-6 (IL-6) and its soluble receptor (sIL-6R) on immature bovine and adult human knee and ankle cartilage, using an in vitro model, and to test the hypothesis that endogenous IL-6 plays a role in proteoglycan loss caused by a combination of injury and TNFalpha. METHODS: Injured or uninjured cartilage disks were incubated with or without TNFalpha and/or IL-6/sIL-6R. Additional samples were preincubated with an IL-6-blocking antibody Fab fragment and subjected to injury and TNFalpha treatment. Treatment effects were assessed by histologic analysis, measurement of glycosaminoglycan (GAG) loss, Western blot to determine proteoglycan degradation, zymography, radiolabeling to determine chondrocyte biosynthesis, and Western blot and enzyme-linked immunosorbent assay to determine chondrocyte production of IL-6. RESULTS: In bovine cartilage samples, injury combined with TNFalpha and IL-6/sIL-6R exposure caused the most severe GAG loss. Findings in human knee and ankle cartilage were strikingly similar to those in bovine samples, although in human ankle tissue, the GAG loss was less severe than that observed in human knee tissue. Without exogenous IL-6/sIL-6R, injury plus TNFalpha exposure up-regulated chondrocyte production of IL-6, but incubation with the IL-6-blocking Fab significantly reduced proteoglycan degradation. CONCLUSION: Our findings indicate that mechanical injury potentiates the catabolic effects of TNFalpha and IL-6/sIL-6R in causing proteoglycan degradation in human and bovine cartilage. The temporal and spatial evolution of degradation suggests the importance of transport of biomolecules, which may be altered by overload injury. The catabolic effects of injury plus TNFalpha appeared partly due to endogenous IL-6, since GAG loss was partially abrogated by an IL-6-blocking Fab.

Oscillatory tension differentially modulates matrix metabolism and cytoskeletal organization in chondrocytes and fibrochondrocytes.

Several modes of mechanical stimulation, including compression, shear, and hydrostatic pressure, have been shown to modulate chondrocyte matrix synthesis, but the effects of mechanical tension have not been widely explored. Since articular cartilage is primarily loaded in compression, tension is not generally viewed as a major contributor to the stress state of healthy tissue. However, injury or attempted repair may cause tension to become more significant. Additionally, fibrocartilaginous tissues experience significant tensile stresses in their normal mechanical environment. In this study we investigated mechanical tension as a means to modulate matrix synthesis and cytoskeletal organization in bovine articular chondrocytes and meniscal fibrochondrocytes (MFCs) in a three-dimensional fibrin construct culture system. Oscillatory tension was applied to constructs at 1.0 Hz and 0-10% displacement variation using a custom device. For nearly all conditions and both cell types, oscillatory tension inhibited matrix synthesis as indicated by 3H-proline and 35S-sulfate incorporation. Additionally, oscillatory tension significantly increased proliferation by chondrocytes but not MFCs. Confocal imaging revealed that all cells initially displayed a rounded morphology, but over time MFCs spontaneously developed a three-dimensional, stellate morphology with numerous projections containing organized cytoskeletal filaments. Interestingly, while unloaded chondrocytes remained rounded, chondrocytes subjected to oscillatory tension developed a similar stellate morphology. Both the biochemical and morphological results of this study have important implications for successfully developing cartilage and fibrocartilage tissue replacements and repair strategies.

The influence of cyclic tension amplitude on chondrocyte matrix synthesis: experimental and finite element analyses.

While not generally viewed as physiologically significant in articular cartilage, substantial tension can develop in fibrocartilage structures and in articular cartilage injuries. This study examined how different amplitudes of cyclic tension influence chondrocyte matrix synthesis. Bovine articular chondrocytes seeded in fibrin gels were loaded continuously for 48 hours at 1.0 Hz with displacements of 5%, 10%, or 20%. Protein and proteoglycan synthesis were measured by (3)H-proline and (35)S-sulfate incorporation, respectively. A poroelastic finite element model of the fibrin gel was developed to determine the strain distributions, hydrostatic pressures, and fluid velocities within the constructs at the various levels of displacement. Compared to unloaded controls, 10% and 20% displacements inhibited proteoglycan synthesis to the same extent, while 5% displacement had no effect. Tensile loading did not significantly affect protein synthesis. The finite element model predicted a wide range of strains and fluid velocities within the region of the gel analyzed for matrix synthesis, and the ranges overlapped for the different levels of displacement. These results indicate that the cyclic tension amplitude influences chondrocyte proteoglycan synthesis and that there may be a threshold in the response.