TGF-B1 activation in human hamstring cells through growth factor binding peptides on polycaprolactone surfaces

The administration of soluble growth factors (GFs) to injured tendons and ligaments (T/L) is known to promote and enhance the healing process. However, the administration of GFs is a complex, expensive and heavily-regulated process and only achieved by employing supraphysiological GF concentrations. In addition, for proper healing, specific and spatial immobilization of the GFs (s) is critical. We hypothesized that biomaterials functionalized with GF-binding peptides can be employed to capture endogenous GFs in a spatially-controlled manner, thus overcoming the need for the exogenous administration of supraphysiological doses of GFs. Here we demonstrate that the modification of films of polycaprolactone (PCL) with transforming growth factor β1 (TGF-β1)-binding peptides allows GFs to be captured and presented to the target cells. Moreover, using a TGF-β reporter cell line and immunocytochemistry, we show that the GFs retained their biological activity. In human primary tendon cells, the immobilized TGF-β1 activated TGF-β target genes ultimately lead to a 2.5-fold increase in total collagen matrix production. In vivo implantation in rats clearly shows an accumulation of TGF-β1 on the polymer films functionalized with the TGF-β1-binding peptide when compared with the native films. This accumulation leads to an increase in the recruitment of inflammatory cells at day 3 and an increase in the fibrogenic response and vascularization around the implant at day 7. The results herein presented will endow current and future medical devices with novel biological properties and by doing so will accelerate T/L healing

Bioactive Tape With BMP-2 Binding Peptides Captures Endogenous Growth Factors and Accelerates Healing After Anterior Cruciate Ligament Reconstruction

Background: The anterior cruciate ligament (ACL) has poor regenerative capacity, and an injury leads to loss of function, limiting quality of life and increasing the incidence of osteoarthritis. Surgical interventions can stabilize the joint and improve functional recovery. The delivery of growth factors (GFs) enhances the healing process; however, this is complex in its regulation, is high in costs, has side effects, and can only be accomplished with supraphysiological concentrations and thus is currently not clinically feasible. However, the immobilization of a patient’s endogenous GFs in biomaterials can overcome these problems. Purpose: To develop a method to capture endogenous bone morphogenetic protein–2 (BMP-2) and ultimately show enhanced ACL healing in vivo using this novel methodology. Study Design: Controlled laboratory study. Methods: BMP-2 binding peptides were synthetized, purified, and immobilized on polycaprolactone (PCL) films. The affinity between the peptide and human BMP-2 (hBMP-2) was confirmed with immunofluorescence and enzyme-linked immunosorbent assay. The C2C12 Luc reporter cell line was used to confirm the bioactivity of immobilized BMP-2. For in vivo experiments, the same functionalization technology was applied to the commercially available Polytape, and the functionalized tape was sutured together with the graft used for ACL reconstruction in rats. Each animal underwent reconstruction with either native Polytape (n = 3) or Polytape with BMP-2 binding peptides (n = 3). At 2 and 6 weeks after surgery, the graft was assessed by histology and micro–computed tomography. Results: The covalent immobilization of the peptide in PCL was successful, allowing the peptide to capture hBMP-2, which remained bioactive and led to the osteogenic differentiation of C2C12. In vivo experiments confirmed the potential of the Polytape functionalized with the BMP-2 binding peptide to capture endogenous BMP-2, leading to enhanced bone formation inside the femoral and tibial tunnels and ultimately improving the graft’s quality. Conclusion: The incorporation of BMP-2 binding peptides into materials used for ACL reconstruction can capture endogenous hBMP-2, which enhances the healing process inside the bone tunnels. Clinical Relevance: These results demonstrate the potential of using synthetic peptides to endow biomaterials with novel biological functions, namely to capture and immobilize endogenous GFs

Respiration from a tropical forest ecosystem: partitioning of sources and low carbon use efficiency

Understanding how tropical forest carbon balance will respond to global change requires knowledge of individual heterotrophic and autotrophic respiratory sources, together with factors that control respiratory variability. We measured leaf, live wood, and soil respiration, along with additional environmental factors over a 1-yr period in a Central Amazon terra firme forest. Scaling these fluxes to the ecosystem, and combining our data with results from other studies, we estimated an average total ecosystem respiration (R-eco) of 7.8 mumol(.)m(-2.)s(-1). Average estimates (per unit ground area) for leaf, wood, soil, total heterotrophic, and total autotrophic respiration were 2.6, 1.1, 3.2, 5.6, and 2.2 mumol(.)m(-2.)s(-1), respectively. Comparing autotrophic respiration with net primary production (NPP) estimates indicated that only similar to30% of carbon assimilated in photosynthesis was used to construct new tissues, with the remaining 70% being respired back to the atmosphere as autotrophic respiration. This low ecosystem carbon use efficiency (CUE) differs considerably from the relatively constant CUE of similar to0.5 found for temperate forests. Our R-eco estimate was comparable to the above-canopy flux (F-ac) from eddy covariance during defined sustained high turbulence conditions (when presumably F-ac = R-eco) of 8.4 (95% CI = 7.59.4). Multiple regression analysis demonstrated that similar to50% of the nighttime variability in Fa, was accounted for by friction velocity (u*, a measure of turbulence) variables. After accounting for u* variability, mean F-ac varied significantly with seasonal and daily changes in precipitation. A seasonal increase in precipitation resulted in a decrease in F-ac similar to our soil respiration response to moisture. The effect of daily changes in precipitation was complex: precipitation after a dry period resulted in a large increase in F-ac whereas additional precipitation after a rainy period had little effect. This response was similar to that of surface litter (coarse and fine), where respiration is greatly reduced when moisture is limiting, but increases markedly and quickly saturates with an increase in moisture

The regenerative effect of different growth factors and platelet lysate on meniscus cells and mesenchymal stromal cells and proof of concept with a functionalized meniscus implant

Meniscus regeneration could be enhanced by targeting meniscus cells and mesenchymal stromal cells (MSCs) with the right growth factors. Combining these growth factors with the Collagen Meniscus Implant (CMI®) could accelerate cell ingrowth and tissue formation in the implant and thereby improve clinical outcomes. Using a transwell migration assay and a micro-wound assay, the effect of insulin-like growth factor-1, platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), transforming growth factor beta 1 (TGF-β1), fibroblast growth factor, and platelet lysate (PL) on migration and proliferation of meniscus cells and MSCs was assessed. The formation of extracellular matrix under influence of the above-mentioned growth factors was assessed after 28 days of culture of both MSCs and meniscus cells. As a proof of concept, the CMI® was functionalized with a VEGF binding peptide and coated with platelet-rich plasma (PRP) for clinical application. Our results demonstrate that PDGF, TGF-β1, and PL stimulate migration, proliferation, and/or extracellular matrix production of meniscus cells and MSCs. Additionally, the CMI® was successfully functionalized with a VEGF binding peptide and PRP which increased migration of meniscus cell and MSC into the implant. This study demonstrates proof of concept of functionalizing the CMI® with growth factor binding peptides. A CMI® functionalized with the right growth factors holds great potential for meniscus replacement after partial meniscectomy

VEGF functionalization of suture tape results in decreased graft inflammatory and catabolic response in a rabbit model of ACL reconstruction

IntroductionImproving ligament reconstruction biology may potentially be achieved through capturing autologous circulating factors such as vascular endothelial growth factor (VEGF) using commercially available biomaterials.ObjectivesTo evaluate anterior cruciate ligament reconstruction (ACLR) using a VEGF functionalized suture tape in a rabbit model of ACLR with a semitendinosus autograft.MethodsVEGF-binding peptides were covalently bonded to polyethylene suture tape (ST) to generate functionalized constructs. Forty-six female New Zealand white rabbit ACLs were reconstructed with semitendinosus hamstring autograft (n = 6), hamstring + ST (n = 16), hamstring + scrambled peptide ST (n = 17), and hamstring + VEGF-functionalized ST (n = 17). Healing was evaluated at 2to 4 weeks using PCR, RNA sequencing, micro CT, histology, and biomechanical testing.ResultsAll rabbits successfully underwent ACLR, with no adverse events. ACLR with VEGF ST demonstrated significant decreases in inflammatory response (CD14, CD163), catabolism (MMP1, MMP3), and apoptosis (TNFSF10, Caspase-10) markers as compared to nonfunctionalized ST (P ≤ .04). µCT demonstrated similar bone tunnel mineral density in hamstring + VEGF ST rabbits as compared to hamstring + scrambled ST controls (P ≥ .31). Histology and biomechanical testing similarly demonstrated no adverse effects of VEGF-based immunomodulation on the tendon grafts.ConclusionsUsing a rabbit model, ACLR performed with VEGF-functionalized suture tape demonstrated significantly decreased markers of inflammation, catabolism, and apoptosis as compared to ACLR with nonfunctionalized suture tape. No adverse effects of functionalization were noted. Future studies should further investigate the utility of functionalized suture tape in ACLR

A Versatile Protocol for Studying Anterior Cruciate Ligament Reconstruction in a Rabbit Model

Anterior cruciate ligament (ACL) injuries are frequent, as >200,000 injuries occur in the United States alone each year. Owing to the risks for associated meniscus and cartilage damage, ACL injuries are a significant source of both orthopedic care and research. Given the extended recovery course after ACL injury, which often lasts 1–2 years, and is associated with limited participation in sports and activities of daily living for patients, there is a critical need for the evolution of new and improved methods for ACL repair. Subsequently, animal models of ACL reconstruction (ACLR) play a key role in the development and initial trialing of novel ACL interventions. This article provides a clear operative description and associated illustrations for a validated, institutional animal care and use committee, and veterinarian approved and facile model of ACLR to serve researchers investigating ACLR

DS_10.1177_0363546518787507 – Supplemental material for Bioactive Tape With BMP-2 Binding Peptides Captures Endogenous Growth Factors and Accelerates Healing After Anterior Cruciate Ligament Reconstruction

<p>Supplemental material, DS_10.1177_0363546518787507 for Bioactive Tape With BMP-2 Binding Peptides Captures Endogenous Growth Factors and Accelerates Healing After Anterior Cruciate Ligament Reconstruction by João F. Crispim, Sai C. Fu, Yuk W. Lee, Hugo A.M. Fernandes, Pascal Jonkheijm, Patrick S.H. Yung and Daniël B.F. Saris in The American Journal of Sports Medicine</p