Enzymatic formation of modular cell-instructive fibrin analogs for tissue engineering

The molecular engineering of cell-instructive artificial extracellular matrices is a powerful means to control cell behavior and enable complex processes of tissue formation and regeneration. This work reports on a novel method to produce such smart biomaterials by recapitulating the crosslinking chemistry and the biomolecular characteristics of the biopolymer fibrin in a synthetic analog. We use activated coagulation transglutaminase factor XIIIa for site-specific coupling of cell adhesion ligands and engineered growth factor proteins to multiarm poly(ethylene glycol) macromers that simultaneously form proteolytically sensitive hydrogel networks in the same enzyme-catalyzed reaction. Growth factor proteins are quantitatively incorporated and released upon cell-derived proteolytic degradation of the gels. Primary stromal cells can invade and proteolytically remodel these networks both in an in vitro and in vivo setting. The synthetic ease and potential to engineer their physicochemical and bioactive characteristics makes these hybrid networks true alternatives for fibrin as provisional drug delivery platforms in tissue engineering.

Engineered fibrin matrices for functional display of cell membrane-bound growth factor-like activities: study of angiogenic signaling by ephrin-B2

With the rapid increase in approaches to pro- or anti-angiogenic therapy, new and effective methodologies for administration of cell-bound growth factors will be required. We sought to develop the natural hydrogel matrix fibrin as platform for extensive interactions and continuous signaling by the vascular morphogen ephrin-B2 that normally resides in the plasma membrane and requires multivalent presentation for ligation and activation of Eph receptors on apposing endothelial cell surfaces. Using fibrin and protein engineering technology to induce multivalent ligand presentation, a recombinant mutant ephrin-B2 receptor binding domain was covalently coupled to fibrin networks at variably high densities. The ability of fibrin-bound ephrin-B2 to act as ligand for endothelial cells was preserved, as demonstrated by a concomitant, dose-dependent increase of endothelial cell binding to engineered ephrin-B2-fibrin substrates in vitro. The therapeutic relevance of ephrin-B2-fibrin implant matrices was demonstrated by a local angiogenic response in the chick embryo chorioallontoic membrane evoked by the local and prolonged presentation of matrix-bound ephrin-B2 to tissue adjacing the implant. This new knowledge on biomimetic fibrin vehicles for precise local delivery of membrane-bound growth factor signals may help to elucidate specific biological growth factor function, and serve as starting point for development of new treatment strategies.

Association of vital exhaustion and depressive symptoms with changes in fibrin D-dimer to acute psychosocial stress

OBJECTIVE: Vital exhaustion and depression are psychosocial risk factors of coronary artery disease. A hypercoagulable state in response to acute psychosocial stress contributes to atherothrombotic events. We aimed to investigate the hypothesis that vital exhaustion and depression correlate with stress-induced changes in the hypercoagulability marker D-dimer. METHODS: Thirty-eight healthy and nonsmoking school teachers (mean age 50+/-8 years, 55% women) completed the nine-item Maastricht Vital Exhaustion Questionnaire and the seven-item depression subscale of the Hospital Anxiety and Depression Scale. Within 1 week, subjects twice underwent the Trier Social Stress Test (i.e., preparation phase, mock job interview, and mental arithmetic that totaled 13 min). Plasma D-dimer levels were determined at five time points during the protocol. RESULTS: Vital exhaustion (P=.022; eta(2)=.080) and depressive symptoms (P=.011; eta(2)=.090) were associated with stress-induced changes in D-dimer levels over time controlling for sex and age. Elevated levels of vital exhaustion (r=-.46, P=.005) and of depression (r=-.51, P=.002) correlated with reduced D-dimer increase from pre-stress to immediately post-stress. Also, elevated vital exhaustion (r=.34, P=.044) and depression (r=.41, P=.013) were associated with increase (i.e., attenuated recovery) of D-dimer levels between 20 and 45 min post-stress. Controlling for stress hormone and blood pressure reactivity did not substantially alter these results. CONCLUSION: The findings suggest an attenuated immediate D-dimer stress response and delayed recovery of D-dimer levels post-stress with elevated vital exhaustion and depressive symptoms. In particular, the prolonged hypercoagulability after stress cessation might contribute to the atherothrombotic risk previously observed with vital exhaustion and depression, even at subclinical levels.

Cell-demanded liberation of VEGF121 from fibrin implants induces local and controlled blood vessel growth

Although vascular endothelial growth factor (VEGF) has been described as a potent angiogenic stimulus, its application in therapy remains difficult: blood vessels formed by exposure to VEGF tend to be malformed and leaky. In nature, the principal form of VEGF possesses a binding site for ECM components that maintain it in the immobilized state until released by local cellular enzymatic activity. In this study, we present an engineered variant form of VEGF, alpha2PI1-8-VEGF121, that mimics this concept of matrix-binding and cell-mediated release by local cell-associated enzymatic activity, working in the surgically-relevant biological matrix fibrin. We show that matrix-conjugated alpha2PI1-8-VEGF121 is protected from clearance, contrary to native VEGF121 mixed into fibrin, which was completely released as a passive diffusive burst. Grafting studies on the embryonic chicken chorioallantoic membrane (CAM) and in adult mice were performed to assess and compare the quantity and quality of neovasculature induced in response to fibrin implants formulated with matrix-bound alpha2PI1-8-VEGF121 or native diffusible VEGF121. Our CAM measurements demonstrated that cell-demanded release of alpha2PI1-8-VEGF121 increases the formation of new arterial and venous branches, whereas exposure to passively released wild-type VEGF121 primarily induced chaotic changes within the capillary plexus. Specifically, our analyses at several levels, from endothelial cell morphology and endothelial interactions with periendothelial cells, to vessel branching and network organization, revealed that alpha2PI1-8-VEGF121 induces vessel formation more potently than native VEGF121 and that those vessels possess more normal morphologies at the light microscopic and ultrastructural level. Permeability studies in mice validated that vessels induced by alpha2PI1-8-VEGF121 do not leak. In conclusion, cell-demanded release of engineered VEGF121 from fibrin implants may present a therapeutically safe and practical modality to induce local angiogenesis.

Increased vascularization during early healing after biologic augmentation in repair of chronic rotator cuff tears using autologous leukocyte- and platelet-rich fibrin (L-PRF): a prospective randomized controlled pilot trial

HYPOTHESIS We hypothesized that arthroscopic rotator cuff repairs using leukocyte- and platelet-rich fibrin (L-PRF) in a standardized, modified protocol is technically feasible and results in a higher vascularization response and watertight healing rate during early healing. METHODS Twenty patients with chronic rotator cuff tears were randomly assigned to 2 treatment groups. In the test group (N = 10), L-PRF was added in between the tendon and the bone during arthroscopic rotator cuff repair. The second group served as control (N = 10). They received the same arthroscopic treatment without the use of L-PRF. We used a double-row tension band technique. Clinical examinations including subjective shoulder value, visual analog scale, Constant, and Simple Shoulder Test scores and measurement of the vascularization with power Doppler ultrasonography were made at 6 and 12 weeks. RESULTS There have been no postoperative complications. At 6 and 12 weeks, there was no significant difference in the clinical scores between the test and the control groups. The mean vascularization index of the surgical tendon-to-bone insertions was always significantly higher in the L-PRF group than in the contralateral healthy shoulders at 6 and 12 weeks (P = .0001). Whereas the L-PRF group showed a higher vascularization compared with the control group at 6 weeks (P = .001), there was no difference after 12 weeks of follow-up (P = .889). Watertight healing was obtained in 89% of the repaired cuffs. DISCUSSION/CONCLUSIONS Arthroscopic rotator cuff repair with the application of L-PRF is technically feasible and yields higher early vascularization. Increased vascularization may potentially predispose to an increased and earlier cellular response and an increased healing rate.

Enhancement of bone healing using non-glycosylated rhBMP-2 released from a fibrin matrix in dogs and cats

OBJECTIVES To test a non-glycosylated recombinant human bone morphogenetic protein-2 (ngly-rhBMP-2)/fibrin composite, which has been shown experimentally to enhance healing of bone defects in rodents, in a clinical case series of dogs and cats undergoing treatment for fracture non-unions and arthrodesis. METHODS A ngly-rhBMP-2/fibrin composite was applied in 41 sites in 38 dogs and cats for which a cancellous bone autograft was indicated, replacing the graft. RESULTS Bridging of the bone defect with functional bone healing was achieved in 90 per cent of the arthrodesis and fracture nonunions treated in this manner. CLINICAL SIGNIFICANCE This prospective clinical study demonstrates the beneficial effects of ngly-rhBMP-2 in a specially designed fibrin matrix on the treatment of bone defects, and validates the use of this composite as an alternative to bone autografts in dogs and cats.

Classification of platelet concentrates (Platelet-Rich Plasma-PRP, Platelet-Rich Fibrin-PRF) for topical and infiltrative use in orthopedic and sports medicine: current consensus, clinical implications and perspectives.

Platelet concentrates for topical and infiltrative use - commonly termed Platetet-Rich Plasma (PRP) or Platelet-Rich Fibrin (PRF) - are used or tested as surgical adjuvants or regenerative medicine preparations in most medical fields, particularly in sports medicine and orthopaedic surgery. Even if these products offer interesting therapeutic perspectives, their clinical relevance is largely debated, as the literature on the topic is often confused and contradictory. The long history of these products was always associated with confusions, mostly related to the lack of consensual terminology, characterization and classification of the many products that were tested in the last 40 years. The current consensus is based on a simple classification system dividing the many products in 4 main families, based on their fibrin architecture and cell content: Pure Platelet-Rich Plasma (P-PRP), such as the PRGF-Endoret technique; Leukocyte- and Platelet-Rich Plasma (LPRP), such as Biomet GPS system; Pure Platelet-Rich Fibrin (P-PRF), such as Fibrinet; Leukocyte- and Platelet-Rich Fibrin (L-PRF), such as Intra-Spin L-PRF. The 4 main families of products present different biological signatures and mechanisms, and obvious differences for clinical applications. This classification serves as a basis for further investigations of the effects of these products. Perspectives of evolutions of this classification and terminology are also discussed, particularly concerning the impact of the cell content, preservation and activation on these products in sports medicine and orthopaedics.

In vitro effects of 3 % hypertonic saline and 20 % mannitol on canine whole blood coagulation and platelet function.

BACKGROUND: Hyperosmolar therapy, using either mannitol or hypertonic saline (HTS), is considered the treatment of choice for intracranial hypertension. However, hyperosmolar agents may impair coagulation and platelet function, limiting their use in patients at risk for hemorrhage. Despite this, studies evaluating the effects of mannitol compared to other hyperosmolar agents in dogs are largely lacking. The aim of this study was to compare the in vitro effects on global hemostasis and platelet function of 20 % mannitol and 3 % HTS on canine blood. METHODS: Citrated whole blood from 15 healthy dogs was diluted with 0.9 % saline, 20 % mannitol and 3 % HTS in ratios of 1:16 and 1:8. Rotational thromboelastometry (ROTEM) was used to assess clotting time (CT), clot formation time (CFT) and maximal clot firmness (MCF) following extrinsic activation (Ex-tem) and after platelet inhibition (Fib-tem). A platelet function analyzer (PFA-100) was used to assess closure time (CtPFA). RESULTS: No significant differences were observed between untreated whole blood and samples diluted with saline. Samples diluted with both mannitol and HTS were hypocoagulable compared to untreated whole blood samples. At a dilution of 1:16, no significant differences were found between any measured parameter in samples diluted with saline compared to mannitol or HTS. At a 1:8 dilution, CtPFA was prolonged in samples diluted with mannitol and HTS compared to saline, and CtPFA was prolonged more with mannitol than HTS. Ex-tem CT was increased at a 1:8 dilution with mannitol compared to HTS. Ex-tem CFT was prolonged at a 1:8 dilution with both agents compared to saline, and was prolonged more with mannitol than HTS. Ex-tem MCF was reduced at a 1:8 dilution with both agents compared to saline. DISCUSSION AND CONCLUSIONS: Data in this study indicate that both mannitol and HTS affect canine platelet function and whole blood coagulation in vitro in a dose-dependent fashion. The most pronounced effects were observed after high dilutions with mannitol, which impaired platelet aggregation, clot formation time, clot strength, and fibrin formation significantly more than HTS. Further in vivo studies are necessary before recommendations can be made

Evaluation of HA-fibrin-based hydrogel for restoration of degenerated intervertebral disc

Introduction: Intervertebral disc degeneration is associated with loss of nucleus pulposus (NP) tissue and reduced disc height[1]. A number of therapies, including synthetic and natural biomaterials, have been developed to restore full disc function and to minimize the pain and disability caused by this disease. Fibrin-based biomaterials are used as a replacement for NP or as a cell carrier for tissue engineering approaches[2]. While the behavior of such gels is well-characterized from a material point of view, little is known about their contribution to intervertebral disc (IVD) restoration under dynamic loads. The aim of the present study is the evaluation of a hyaluronic acid fibrin-based hydrogel (ProCore) used to repair an in vitro model of disc degeneration under dynamic loading. Methods: In vitro model of disc degeneration was induced in intact coccygeal bovine IVD by papain digestion of the NP as previously described[3]. In order to characterize fibrin hydrogels, four experimental groups were considered: 1) intact IVD (control), 2) IVD injected with PBS, 3) injection of hydrogels in degenerative IVD and 4) injection of hydrogels in combination with human bone marrow-derived mesenchymal stem cells (MSC) in degenerative IVD. All of the groups were subjected to dynamic loading protocols consisting of 0.2MPa static compression superimposed with ±2° torsion at 0.2Hz for 8h per day and maintained for 7 days. Additionally, one group consisted of degenerative IVD injected with hydrogel and subjected to static compression. Disc heights were monitored after the duration of the loading and compared to the initial disc height. The macrostructure of the formed tissue and the cellular distribution was evaluated by histological means. Results: After one week of loading, the degenerative IVD filled with hydrogel in combination with MSC (dynamic load), hydrogels (dynamic load) and hydrogels (static load) showed a reduction in height by 30%, 15% and 20%, respectively, as compared to their initial disc height. Histological sections showed that the HA-fibrin gel fully occupied the nucleotomized region of the disc and that fibrin was effective in filling the discontinuities of the cavity region. Furthermore, the cells were homogenously distributed along the fibrin hydrogels after 7 days of loading. Discussion: In this study, we showed that fibrin hydrogels showed a good integration within the papain-induced model of disc degeneration and can withstand the applied loads. Fibrin hydrogels can contribute to disc restoration by possibly maintaining adequate stiffness of the tissue and thus preventing disorganization of the surrounding IVD. References: 1. Jarman, J.P., Arpinar, V.E., Baruah, D., Klein, A.P., Maiman, D.J., and Tugan Muftuler, L. (2014). Intervertebral disc height loss demonstrates the threshold of major pathological changes during degeneration. Eur Spine J . 2. Colombini, A., Ceriani, C., Banfi, G., Brayda-Bruno, M., and Moretti, M. (2014). Fibrin in intervertebral disc tissue engineering. Tissue Eng Part B Rev . 3. Chan, S.C., Bürki, A., Bonél, H.M., Benneker, L.M., and Gantenbein-Ritter, B. (2013). Papain-induced in vitro disc degeneration model for the study of injectable nucleus pulposus therapy. Spine J 13, 273-283. Acknowledgement We thank the Swiss National Science Foundation SNF #310030_153411 for funding.

Intervertebral Disc Repair using Fleece-Membrane Composite Silk Material and Genipin-enhanced Fibrin Hydrogel

Introduction: Treating low back pain (LBP) has become an increasing challenge, as it is one of the main factors causing pain and is accompanied by high costs for the individual and the society. LBP can be caused by trauma of the intervertebral disc (IVD) or IVD degeneration. In the case of disc herniation the inner gelatinous part of the IVD, called nucleus pulposus, is pressed through the fibrous, annulus fibrosus that forms the outer part of the IVD. Today’s gold standard for treatment is extensive surgery as removal of the IVD and fusion of the vertebrae. In order to find a more gentle way to treat LBP and restore the native IVD we use a novel silk fleece-membrane composite from genetically modified silk worms whose silk contains a growth factor (GDF-6) that is associated with pushing stem cells towards a disc like phenotype (1). By combining it with a genipin-enhanced fibrin hydrogel we tested its suitability in organ culture on prior injured bovine IVD in our custom built two-degree of freedom bioreactor to mimic natural loading conditions. Material & Methods: Bovine IVDs of 12-17 months old animals were isolated by first removing all surrounding tissue followed by cutting out the IVDs as previously described (2). Culturing of discs occurred in high glucose Dulbecco's Modified Eagle Medium (HG-DMEM) supplemented with 5% serum as previously described (2). On the next day injury was induced using a 2mm biopsy punch (Polymed, Switzerland). The formed cavity was filled with (0.4%) genipin-enhanced human based fibrin hydrogel (35-55mg/mL human fibrinogen, Baxter, Austria) and sealed with a silk fleece-membrane composite (Spintec Engineering, Germany). Different culture conditions were applied: free swelling, static diurnal load of 0.2MPa for 8h/d and complex loading at 0.2MPa compression combined with ± 2° torsion at 0.2Hz for 8h/d (2). After 14 days of culture cell activity was determined with resazurin assay. Additionally, glycosaminoglycan (dimethyl-methylene blue), DNA (Hoechst) and collagen content (hydroxy- proline) were determined. Finally, real-time qPCR of major IVD marker and inflammation genes was performed to judge integrity of IVDs. Results: The fibrin hydrogel is able to keep the silk seal in place throughout the 14 days of in organ culture under all conditions. Additionally, cell activity showed optimistic results and we could not confirm negative effects of the repaired discs regarding overexpression of inflammation markers. Conclusions: The genipin-enhanced fibrin hydrogel in combination with the silk fleece- membrane composite seems to be a promising approach for IVD repair. Currently we assess the capability of GDF-6 incorporated in our silk composites on human mesenchymal stem cells and later on in organ culture. References 1. Clarke LE, McConnell JC, Sherratt MJ, Derby B, Richardson SM, Hoyland JA. Growth differentiation factor 6 and transforming growth factor-beta differentially mediate mesenchymal stem cell differentiation, composition and micromechanical properties of nucleus pulposus constructs. Arthritis Res Ther 2014, Mar 12;16(2):R67. 2. Chan SC, Gantenbein-Ritter B. Preparation of intact bovine tail intervertebral discs for organ culture. J Vis Exp 2012, Feb 2;60(60):e3490. Acknowledgements. This work is funded by the Gebert Rüf Foundation, project number GRS-028/13.

Platelet-rich concentrates differentially release growth factors and induce cell migration in vitro

BACKGROUND Platelet-rich concentrates are used as a source of growth factors to improve the healing process. The diverse preparation protocols and the gaps in knowledge of their biological properties complicate the interpretation of clinical results. QUESTIONS/PURPOSES In this study we aimed to (1) analyze the concentration and kinetics of growth factors released from leukocyte- and platelet-rich fibrin (L-PRF), leukocyte- and platelet-rich plasma (L-PRP), and natural blood clot during in vitro culture; (2) investigate the migration of mesenchymal stem cells (MSCs) and human umbilical vein endothelial cells (HUVECs) as a functional response to the factors released; and (3) uncover correlations between individual growth factors with the initial platelet/leukocyte counts or the induced cell migration. METHODS L-PRF, L-PRP, and natural blood clot prepared from 11 donors were cultured in vitro for 28 days and media supernatants collected after 8 hours and 1, 3, 7, 14, and 28 days. Released transforming growth factor β1 (TGF-β1), vascular endothelial growth factor (VEGF), insulin growth factor (IGF-1), platelet-derived growth factor AB (PDGF-AB), and interleukin-1β (IL-1β) were measured in the supernatants with enzyme-linked immunosorbent assay. Migration of MSC and HUVEC induced by the supernatants was evaluated in Boyden chambers. RESULTS More TGF-ß1 was released (mean ± SD in pg/mL of blood) from L-PRF (37,796 ± 5492) compared with L-PRP (23,738 ± 6848; p < 0.001) and blood clot (3739 ± 4690; p < 0.001), whereas more VEGF and IL-1ß were released from blood clot (1933 ± 704 and 2053 ± 908, respectively) compared with both L-PRP (642 ± 208; p < 0.001 and 273 ± 386; p < 0.001, respectively) and L-PRF (852 ± 376; p < 0.001 and 65 ± 56, p < 0.001, respectively). No differences were observed in IGF-1 and PDGF-AB released from any of the concentrates. TGF-β1 release peaked at Day 7 in L-PRF and at 8 hours and Day 7 in L-PRP and 8 hours and Day 14 in blood clot. In all concentrates, main release of VEGF occurred between 3 and 7 days and of IL-1β between Days 1 and 7. IGF-1 and PDGF-AB were released until Day 1 in L-PRP and blood clot, in contrast to sustained release over the first 3 days in L-PRF. The strongest migration of MSC occurred in response to L-PRF, and more HUVEC migration was seen in L-PRF and blood clot compared with L-PRP. TGF-β1 correlated with initial platelet counts in L-PRF (Pearson r = 0.66, p = 0.0273) and initial leukocyte counts in L-PRP (Pearson r = 0.83, p = 0.0016). A positive correlation of IL-1β on migration of MSC and HUVEC was revealed (Pearson r = 0.16, p = 0.0208; Pearson r = 0.31, p < 0.001). CONCLUSIONS In comparison to L-PRP, L-PRF had higher amounts of released TGF-β1, a long-term release of growth factors, and stronger induction of cell migration. Future preclinical studies should confirm these data in a defined injury model. CLINICAL RELEVANCE By characterizing the biologic properties of different platelet concentrates in vitro, we may gain a better understanding of their clinical effects and develop guidelines for specific future applications.