Functionalizing Fibrin Hydrogels with Thermally Responsive Oligonucleotide Tethers for On-Demand Delivery.

There are a limited number of stimuli-responsive biomaterials that are capable of delivering customizable dosages of a therapeutic at a specific location and time. This is especially true in tissue engineering and regenerative medicine applications, where it may be desirable for the stimuli-responsive biomaterial to also serve as a scaffolding material. Therefore, the purpose of this study was to engineer a traditionally non-stimuli responsive scaffold biomaterial to be thermally responsive so it could be used for on-demand drug delivery applications. Fibrin hydrogels are frequently used for tissue engineering and regenerative medicine applications, and they were functionalized with thermally labile oligonucleotide tethers using peptides from substrates for factor XIII (FXIII). The alpha 2-plasmin inhibitor peptide had the greatest incorporation efficiency out of the FXIII substrate peptides studied, and conjugates of the peptide and oligonucleotide tethers were successfully incorporated into fibrin hydrogels via enzymatic activity. Single-strand complement oligo with either a fluorophore model drug or platelet-derived growth factor-BB (PDGF-BB) could be released on demand via temperature increases. These results demonstrate a strategy that can be used to functionalize traditionally non-stimuli responsive biomaterials suitable for on-demand drug delivery systems (DDS).

Keratinocyte Migration in a Three-Dimensional In Vitro Wound Healing Model Co-Cultured with Fibroblasts.

BACKGROUND: Because three-dimensional (3D) models more closely mimic native tissues, one of the goals of 3D in vitro tissue models is to aid in the development and toxicity screening of new drug therapies. In this study, a 3D skin wound healing model comprising of a collagen type I construct with fibrin-filled defects was developed. METHODS: Optical imaging was used to measure keratinocyte migration in the presence of fibroblasts over 7 days onto the fibrin-filled defects. Additionally, cell viability and growth of fibroblasts and keratinocytes was measured using the alamarBlue® assay and changes in the mechanical stiffness of the 3D construct was monitored using compressive indentation testing. RESULTS: Keratinocyte migration rate was significantly increased in the presence of fibroblasts with the cells reaching the center of the defect as early as day 3 in the co-culture constructs compared to day 7 for the control keratinocyte monoculture constructs. Additionally, constructs with the greatest rate of keratinocyte migration had reduced cell growth. When fibroblasts were cultured alone in the wound healing construct, there was a 1.3 to 3.4-fold increase in cell growth and a 1.2 to 1.4-fold increase in cell growth for keratinocyte monocultures. However, co-culture constructs exhibited no significant growth over 7 days. Finally, mechanical testing showed that fibroblasts and keratinocytes had varying effects on matrix stiffness with fibroblasts degrading the constructs while keratinocytes increased the construct's stiffness. CONCLUSION: This 3D in vitro wound healing model is a step towards developing a mimetic construct that recapitulates the complex microenvironment of healing wounds and could aid in the early studies of novel therapeutics that promote migration and proliferation of epithelial cells.

Mesenchymal stem cell growth on and mechanical properties of fibrin-based biomimetic bone scaffolds.

Using the microenvironment of healing bone tissue as inspiration, this study utilized fibrin hydrogels combined with collagen type I and calcium phosphate ceramics to create a biomimetic bone scaffold. The contribution each component had on the growth of mesenchymal stem cells (hMSC) was assessed, and changes in the scaffold's mechanical properties were measured by indentation testing. The results show cell growth was greatest in scaffolds with lower concentrations of fibrinogen complex and followed a similar trend with the addition of collagen. However, cell growth was greatest in fibrin scaffolds with high concentrations of fibrinogen complex when combined with hydroxyapatite-ß-tricalcium phosphate. The fibrin scaffold's stiffness does not significantly change over time, but the addition of collagen to scaffolds with low concentrations of fibrinogen complex had significant increases in stiffness by day 14. These results demonstrate that hMSC do not rapidly degrade fibrin and fibrin-collagen scaffolds in vitro. The data reported here can aid in the design and fabrication of fibrin-based engineered tissues and cell delivery vehicles that promote hMSC growth and viability as well as meet the mechanical requirements of native tissues. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2945-2953, 2016.

Enhancing Tissue Engineering and Regenerative Medicine Product Commercialization: The Role of Science in Regulatory Decision-Making for the TE/RM Product Development.

TERMIS-AM Industry Committee (TERMIS-AM/IC), in collaboration with the TERMIS-Europe (EU)/IC, conducted a symposium involving the European Medicines Agency and the U.S. Food and Drug Administration (FDA) toward building an understanding of the rational basis for regulatory decision-making and providing a framework for decisions made during the evaluation of safety and efficacy of TE/RM technologies. This symposium was held in August 2012 during the TERMIS-WC in Vienna, Austria. Emerging from this international initiative by the European Union and the United States, representatives from the respective agencies demonstrated that there are ongoing interagency efforts for developing common national practices toward harmonization of regulatory requirements for the TE/RM products. To extend a broad-based understanding of the role of science in regulatory decision-making, TERMIS-AM/IC, in cooperation with the FDA, organized a symposium at the 2014 TERMIS-AM Annual Meeting, which was held in Washington, DC. This event provided insights from leaders in the FDA and TERMIS on the current status of regulatory approaches for the approved TE/RM products, the use of science in making regulatory decisions, and TE/RM technologies that are in the development pipeline to address unmet medical needs. A far-ranging discussion with FDA representatives, industrialists, physicians, regenerative medicine biologists, and tissue engineers considered the gaps in today's scientific and regulatory understanding of TE/RM technologies. The identified gaps represent significant opportunities to advance TE/RM technologies toward commercialization.

Concentration of fibrin and presence of plasminogen affect proliferation, fibrinolytic activity, and morphology of human fibroblasts and keratinocytes in 3D fibrin constructs.

Fibrin is a hemostatic protein found in the clotting cascade. It is used in the operating room to stop bleeding and deliver cells and growth factors to heal wounds. However, formulations of clinically approved fibrin are optimized for hemostasis, and the extent to which biochemical and physical cues in fibrin mediate skin cell behavior is not fully understood nor utilized in the design of biomaterials. To determine if the concentration of fibrinogen and the presence of plasminogen affect cell behavior relevant to wound healing, we fabricated three-dimensional fibrin constructs made from 5, 10, or 20 mg/mL of clinical fibrin or plasminogen-depleted (PD) fibrin. We cultured dermal fibroblasts or epidermal keratinocytes in these constructs. Fibroblasts proliferated similarly in both types of fibrin, but keratinocytes proliferated more in low concentrations of clinical fibrin and less in PD fibrin. Clinical fibrin constructs with fibroblasts were less stiff and degraded faster than PD fibrin constructs with fibroblasts. Similarly, keratinocytes degraded clinical fibrin, but not PD fibrin. Fibroblast spreading varied with fibrin concentration in both types of fibrin. In conclusion, the concentration of fibrinogen and the presence of plasminogen affect fibroblast and keratinocyte proliferation, morphology, and fibrin degradation. Creating materials with heterogeneous regions of fibrin formulations and concentrations could be a novel strategy for controlling the phenotype of encapsulated fibroblasts and keratinocytes, and the subsequent biomechanical properties of the construct. However, other well-investigated aspects of wound healing remain to be utilized in the design of fibrin biomaterials, such as autocrine and paracrine signaling between fibroblasts, keratinocytes, and immune cells.

Awareness of the role of science in the FDA regulatory submission process: a survey of the TERMIS-Americas membership.

The Industry Committee of the Tissue Engineering Regenerative Medicine International Society, Americas Chapter (TERMIS-AM) administered a survey to its membership in 2013 to assess the awareness of science requirements in the U.S. Food and Drug Administration (FDA) regulatory process. One hundred forty-four members responded to the survey. Their occupational and geographical representation was representative of the TERMIS-AM membership as a whole. The survey elicited basic demographic information, the degree to which members were involved in tissue engineering technology development, and their plans for future involvement in such development. The survey then assessed the awareness of general FDA scientific practices as well as specific science requirements for regulatory submissions to the Center for Biologics Evaluation and Research (CBER), the Center for Drug Evaluation and Research (CDER), the Center for Devices and Radiological Health (CDRH), and the Office of Combination Projects (OCP). The FDA-specific questions in the survey were culled from guidance documents posted on the FDA web site ( www.fda.gov ). One of the answer options was an opt-out clause that enabled survey respondents to claim a lack of sufficient awareness of the topic to answer the question. This enabled the stratification of respondents on the basis of confidence in the topic. Results indicate that across all occupational groups (academic, business, and government) that are represented in the TERMIS-AM membership, the awareness of FDA science requirements varies markedly. Those who performed best were for-profit company employees, consultants, and government employees; while students, professors, and respondents from outside the USA performed least well. Confidence in question topics was associated with increased correctness in responses across all groups, though the association between confidence and the ability to answer correctly was poorest among students and professors. Though 80% of respondents claimed involvement in the development of a tissue engineering technology, their responses were no more correct than those who were not. Among those developing tissue engineering technologies, few are taking advantage of existing standards organizations to strengthen their regulatory submissions. The data suggest that early exposure to regulatory experts would be of value for those seeking to bring their technology to the market. For all groups studied but especially for students and professors, formal initial or continuing education in Regulatory Science should be considered to best support translational tissue engineering research and development. In addition, the involvement of standards development organizations during tissue engineering technology development is strongly recommended.

The effect of fibrinogen, collagen type I, and fibronectin on mesenchymal stem cell growth and differentiation into osteoblasts.

We have shown that human mesenchymal stem cells (hMSCs) have the potential to differentiate into bone when seeded within three-dimensional (3-D) fibrin constructs. Proteins endogenous to the fibrin construct and those secreted by cells in the 3-D constructs provide cues that can promote differentiation of hMSCs along with mechanical support for cell growth and migration. In this study, we decided to take a step back and examine the effect different extracellular matrix (ECM) proteins--fibrinogen, fibronectin, and collagen type I--had on hMSC osteogenic differentiation on two-dimensional (2-D) monolayer cultures. Briefly, 24-well tissue culture plates pre-coated with either fibrinogen (10 mg/mL), fibronectin (10 µg/mL), or collagen type I (1 mg/mL) were seeded with 25,000 cells/well and cultured in normal growth medium or in osteogenic induction medium. At days 1, 7, 14, 21, and 30, cultures were assessed for cell growth using alamarBlue(®) and osteogenic indicators using alkaline phosphatase and Von Kossa staining. The results show that collagen type I stained positive for calcium deposition the greatest by day 30 in both osteogenic medium and standard culture medium. However, fibrinogen had the greatest staining in osteogenic medium at day 21 and fibronectin was the only substrate to promote calcium deposition in standard culture medium at day 21. These results suggest that the osteogenic differentiation of hMSCs is influenced by both culturing conditions and substrate and that together they have a synergistic effect. By knowing the effect ECM proteins in 3-D fibrin construct have on promoting osteogenic differentiation of hMSCs, the fabrication of complex, biomimetic models designed to manipulate hMSC differentiation toward an osteoblastic lineage will be improved.

Hurdles in tissue engineering/regenerative medicine product commercialization: a pilot survey of governmental funding agencies and the financial industry.

The Tissue Engineering and Regenerative Medicine International Society of the Americas (TERMIS-AM) Industry Committee conducted a semiquantitative opinion survey in 2010 to delineate potential hurdles to commercialization perceived by the TERMIS constituency groups that participate in the stream of technology commercialization (academia, start-up companies, development-stage companies, and established companies). A significant hurdle identified consistently by each group was access to capital for advancing potential technologies into development pathways leading to commercialization. A follow-on survey was developed by the TERMIS-AM Industry Committee to evaluate the financial industry's perspectives on investing in regenerative medical technologies. The survey, composed of 15 questions, was developed and provided to 37 investment organizations in one of three sectors (governmental, private, and public investors). The survey was anonymous and confidential with sector designation the only identifying feature of each respondent's organization. Approximately 80% of the survey was composed of respondents from the public (n=14) and private (n=15) sectors. Each respondent represents one investment organization with the potential of multiple participants participating to form the organization's response. The remaining organizations represented governmental agencies (n=8). Results from this survey indicate that a high percentage (<60%) of respondents (governmental, private, and public) were willing to invest >$2MM into regenerative medical companies at the different stages of a company's life cycle. Investors recognized major hurdles to this emerging industry, including regulatory pathway, clinical translation, and reimbursement of these new products. Investments in regenerative technologies have been cyclical over the past 10-15 years, but investors recognized a 1-5-year investment period before the exit via Merger and Acquisition (M&A). Investors considered musculoskeletal products and their top technology choice with companies in the clinical stage of development being the most preferred investment targets. All sectors indicated a limited interest in early-stage start-up companies potentially explaining why start-up companies have struggled to access to capital and investors based their investment on the stage of a company's life cycle, reflecting each sector's risk tolerance, exit strategy, time of holding an investment, and investment strategy priorities. Investors highlighted the limited number of regenerative medical companies that have achieved commercial status as a basis for why public investors have been approached by so few companies. Based on respondents to this survey, regenerative medical sponsors seeking capital from the financial industry must keep the explanation of their technology simple, since all sectors considered regenerative medical technology as difficult to evaluate. This survey's results indicate that under the current financial environment, many regenerative medical companies must consider codevelopment or even M&A as nondilutive means of raising capital. The overall summary for this survey highlights the highly varied goals and motivations for the various sectors of the government and financial industries.

Permeability of three-dimensional fibrin constructs corresponds to fibrinogen and thrombin concentrations.

Research in the last few years have focused on the use of three-dimensional (3D) fibrin construct to deliver growth factors and cells. Three-dimensional construct permeability and porosity are important aspects for proper nutrient uptake, gas exchange, and waste removal-factors that are critical for cell growth and survival. We have previously reported that the mechanical strength (stiffness) of 3D fibrin constructs is dependent on the fibrinogen and thrombin concentration. In this study, we established two new in vitro models to examine how fibrin composition affects the final 3D fibrin construct permeability and pore size; thereby, influencing the diffusivity of macromolecules throughout the network of fibrin fibrils. Flow measurements of both liquid and fluoresceinated-dextran microparticles are conducted to calculate the permeability and pore size of 3D fibrin constructs of different fibrinogen and thrombin concentrations. Similarly, the diffusivity of liquid and fluoresceinated-dextran microparticles through these 3D fibrin constructs are determined through diffusion models. Data from these studies show that the structural permeability and pore size of 3D fibrin constructs directly correlate to fibrinogen and thrombin concentration in the final 3D fibrin construct. More specifically, at a constant thrombin concentration of 2 or 5 µ/mL, pore size of the 3D fibrin constructs is dependent on fibrinogen if the concentration is 5 mg/mL and to a lesser extent if the concentration is 10-15 mg/mL. These findings suggest that fibrin's diffusive property can be manipulated to fabricate 3D constructs that are optimized for cellular growth, protein transport, and for the controlled delivery of bioactive molecules such as growth factors.

Proliferation of human keratinocytes and cocultured human keratinocytes and fibroblasts in three-dimensional fibrin constructs.

Over the last several years, our in vitro and in vivo studies have focused on optimizing the use of fibrin to deliver cells. We have shown that some three-dimensional (3D) fibrin constructs with specific fibrinogen and thrombin concentration support robust proliferation of normal human dermal fibroblasts, whereas different fibrinogen and thrombin concentrations support high mesenchymal stem cell proliferation in 3D fibrin constructs. In this article, we found that normal human epithelial keratinocytes proliferate well in 3D fibrin constructs consist of fibrinogen concentration ranging from 17 to 33 mg/mL and thrombin concentration of 1 U/mL. Further, using a new proliferation assay, we studied the proliferation of fibroblasts and keratinocytes cocultured in various 3D fibrin constructs of different fibrinogen and thrombin concentrations. We found that 3D fibrin constructs with a range of fibrinogen concentration (5-34 mg/mL) and a thrombin concentration of 1 U/mL produce an optimal cell proliferation for both cell types when cocultured. This profile of proliferation is different from that seen when keratinocytes or fibroblasts are incorporated separately in 3D fibrin constructs. In conclusion, we found that one needs to choose the fibrinogen and thrombin concentration carefully depending on the cell type to deliver; that is, different fibrin constructs with different fibrinogen and thrombin concentration are required to deliver fibroblasts or keratinocytes alone or to codeliver both cell types. Moreover, there seems to be a cross-talk between keratinocytes and fibroblasts when they are cointroduced in 3D fibrin constructs. This feedback could be due to the effects of growth factors produced by the two cell types in the 3D fibrin constructs.

Hurdles in tissue engineering/regenerative medicine product commercialization: a survey of North American academia and industry.

The Tissue Engineering and Regenerative Medicine International Society-North America (TERMIS-NA) Industry Committee was formed in February 2009 to address the common roadblocks (i.e., hurdles) in the commercialization of tissue engineering/regenerative medicine products for its members. A semiquantitative online opinion survey instrument that delineated potentially sensitive hurdles to commercialization in each of the TERMIS constituency groups that generally participate in the stream of technology commercialization (academia, startup companies, development-stage companies, and established companies) was developed. The survey was opened to each of the 863 members of TERMIS-NA for a period of 5 weeks from October to November 2009. By its conclusion, 215 members (25%) had responded. Their proportionate numbers were closely representative of TERMIS-NA constituencies. The resulting data delineate what each group considers to be its most difficult and also its easiest hurdles in taking a technology to full product development. In addition, each group ranked its perception of the difficult and easy hurdles for all other groups, enabling an assessment of the degree of understanding between groups. The data depict not only critical hurdles in the path to commercialization at each stage in product development but also a variable understanding of perceptions of hurdles between groups. This assessment has provided the Industry Committee with activity foci needed to assist individual groups in the technology-commercialization stream. Moreover, the analysis suggests that enhanced communication between groups engaged in commercialization will be critical to the successful development of products in the tissue engineering/regenerative medicine sector.

Specific fibrinogen and thrombin concentrations promote neuronal rather than glial growth when primary neural cells are seeded within plasma-derived fibrin gels.

Fibrin gels are attractive scaffolds useful for neural tissue engineering applications. The objective of this work was to investigate the apoptotic activity, survival, proliferation, and differentiation of a mixed population of primary neural cells composed of neurons and multipotent precursor cells when cultured in fibrin gels prepared with varying concentrations of fibrinogen (5-25 mg/mL fibrinogen) and thrombin (1-125 U/mL thrombin). Within all fibrin gel formulations tested, the level of apoptosis on day 1 was low and cell survival was equivalent to levels in monolayer culture (67%). Proliferation in gels made from 5 to 12.5 mg/mL fibrinogen was also similar to that observed in monolayer culture, though a lower proliferative response was observed in 25 mg/mL fibrinogen formulations. Relative to monolayer culture, cholinergic and dopaminergic neuronal presence was enhanced, whereas glial cell growth was reduced in fibrin gel cultures. The extent to which levels were altered depended on fibrinogen and thrombin concentration. The findings here suggest the importance of fibrinogen and thrombin concentration in differentially regulating the growth and composition of neural cell populations and are of importance for neural tissue engineering strategies focused on the development of implantable scaffolds for treating neurodegenerative disorders.

Fibrin sealant combined with fibroblasts and platelet-derived growth factor enhance wound healing in excisional wounds.

We test the hypothesis that the fibrinogen-thrombin formulation of fibrin sealant combined with fibroblasts and PDGF-BB enhance cutaneous wound healing. Four formulations varying in fibrinogen and thrombin concentration were applied to full-thickness biopsy wounds in the rabbit ear cutaneous wound healing model with or without cultured rabbit dermal fibroblasts (RDFs; 3 x 10(5) cells/wound) embedded in the fibrinogen component. At post-wounding day 7, there was no difference in the diluted vs. non-diluted formulations for either the promotion of granulation tissue coverage of the open wounds or total granulation tissue area when tested without embedded cells. Including the RDFs, the highest degree of wound coverage by granulation tissue was observed in the combined dilution formulation (17.3 mg/mL fibrinogen, 167 U/mL thrombin; n=10 wounds) that was 167% (p<0.05) of the nondiluted FS containing cells (50 mg/mL fibrinogen, 250 U/mL thrombin; n=10 wounds). Inclusion of fibroblasts increased granulation tissue area within the wounds vs. FS alone (p<0.05) for each diluted formulation although no differences in this parameter were observed within each group (FS alone or with embedded cells). However, addition of the vulnerary growth factor PDGF-BB (3 mg; n=4) with the embedded RDFs in the combined dilution formulation increased granulation tissue area over two-fold (p<0.01) over FS alone. Additionally, the presence of the RDFs promoted incorporation of the granulation tissue with and epithelial migration over the FS suggesting an active interaction between cells delivered to the wound by FS and the host repair cells. The findings suggest the progress of cutaneous defect repair can be enhanced by ex vivo cell delivery in fibrin sealant.

Modulation of 3D fibrin matrix stiffness by intrinsic fibrinogen-thrombin compositions and by extrinsic cellular activity.

Fibrin is a substance formed through catalytic conversion of coagulation constituents: fibrinogen and thrombin. The kinetics of the two constituents determines the structural properties of the fibrin architecture. We have shown previously that changing the fibrinogen and thrombin concentrations in the final three-dimensional (3D) fibrin matrix influenced cell proliferation and differentiation. In this study, we further examined the effect of changing fibrinogen and thrombin concentrations in the absence or presence of fibroblasts on the structural modulus or stiffness of 3D fibrin matrices. We have prepared fibroblast-free and fibroblast-embedded 3D fibrin matrices of different fibrinogen and thrombin formulations, and tested the stiffness of these constructs using standard mechanical testing assays. Results showed that there was a corresponding increase in stiffness with increasing thrombin and fibrinogen concentrations; the increase was more notable with fibrinogen and to a lesser degree with thrombin. The effect of fibroblasts on the stiffness of the fibrin construct was also examined. We have observed a small increase in the stiffness of the fibroblast-incorporated fibrin construct as they proliferated and exhibited spreading morphology. To our knowledge, this is the first comprehensive report detailing the relationship between fibrinogen and thrombin concentrations, cell proliferation, and stiffness in 3D fibrin matrices. The data obtained may lead to optimally design suitable bioscaffolds where we can control both cell proliferation and structural integrity for a variety of tissue engineering applications.

Fibrin as a delivery vehicle for active macrophage activator lipoprotein-2 peptide: in vitro studies.

Fibrin sealants have been used in hemostasis and tissue sealing for over 25 years and recent studies have shown them to be an ideal delivery vehicle for cells and bioactive substances. We examined the use of fibrin as a delivery vehicle for the macrophage activator lipoprotein peptide (MALP)-2. MALP-2, secreted by mycoplasma, plays an important role in an early influx of leukocytes and infiltration by monocytes and their subsequent activation into macrophages as detected by their secretion of cytokines and chemoattractants. We first showed that MALP-2 activated several monocytic cell lines by increasing the expression of cytokines and chemoattractants in these cells. Furthermore, using a reverse transcription-polymerase chain reaction approach, we found that MALP-2 affected the gene expression of its own receptors: TLR2 and TLR4 in various cell types including fibroblasts, keratinocytes, and endothelial cells. Furthermore, the conditioned medium, containing secreted cytokines and chemoattractants, collected from monocytes treated with MALP-2 enhanced fibroblast migration using a standard wound culture assay. Next, we examined MALP-2's effect on the human monocyte cell line when it is mixed with fibrin. Monocytes seeded on three-dimensional fibrin containing MALP-2 secreted more cytokines such as interleukin-6, tumor necrosis factor-alpha, and chemoattractants such as macrophage inflammatory protein 1 alpha and monocyte chemoattractant protein 1 when compared with monocytes seeded on three-dimensional fibrin in the absence of MALP-2. This study supports the use of fibrin to deliver MALP-2, and possibly other peptides, in an active form that might enhance wound healing.

Human mesenchymal stem cell proliferation and osteogenic differentiation in fibrin gels in vitro.

This study analyzed human mesenchymal stem cell (hMSC) behavior in a fibrin sealant. hMSC morphology, proliferation, and osteogenic differentiation were analyzed after up to 28 days of incubation in eight different formulations of fibrin gels (Tisseel) prepared with various concentrations of fibrinogen complex (FC) and thrombin. Cell morphology and distribution within the gels were observed by fluorescence microscopy after cell staining with calcein dye. Cell proliferation was assessed by measuring the fluorescence intensity of the cell suspension stained with calcein dye after dissolution of the gels. A standard alkaline phosphatase (ALP) assay, von Kossa staining, and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) were used to analyze hMSC osteogenic differentiation. Cell behavior varied depending on the gel formulation. Proliferation was higher in the formulations containing a low FC concentration, but ALP activity was higher in the formulations containing a high FC concentration. Variations in thrombin concentration had a lesser effect. Small nodules of mineralization were observed at days 21 and 28 in a formulation containing a high FC concentration, in addition to a marked increase in bone sialoprotein (BSP) gene expression level as well as a lower increase in ALP and osteopontin (OPN) levels. However, there was no significant increase in osteocalcin (OCN) expression, a late marker of osteogenic differentiation, up to day 28. In conclusion, this study demonstrated that hMSC morphology, proliferation, and osteogenic differentiation in fibrin gels depended on the FC/thrombin ratio. hMSCs appeared to undergo osteogenic differentiation when seeded in Tisseel fibrin sealant containing a high FC concentration, but they did not fully differentiate into mature osteoblasts.

The behavior of human mesenchymal stem cells in 3D fibrin clots: dependence on fibrinogen concentration and clot structure.

Natural biopolymers such as human fibrin are appealing to tissue engineers, because fibrin is biocompatible, bioresorbable, and essential in normal wound healing. There have been numerous studies to date to develop a fibrin-based injectable cell delivery system, albeit with varying success. We propose that the outcome of fibrin cell delivery can, in part, be attributed to the relative concentrations of fibrinogen and thrombin solutions (i.e., formulations) and the structure of the final 3D fibrin clot. Formulation-dependent proliferation of human mesenchymal stem cells (hMSCs) within 3D fibrin clots was investigated in vitro. Our results indicate that hMSCs are viable in all fibrin sealant formulations investigated, and proliferation rates vary with fibrin formulations. Furthermore, the fibrinogen solution, not thrombin, was found to have a more dominant role on hMSC proliferation, with dilute fibrinogen solutions promoting greater hMSC proliferation. Confocal and electron microscopy reveal formulation dependence on 3D fibrin clot structure, with dilute fibrinogen solutions yielding more open, homogeneous microstructures. This study suggests that the concentrations of fibrinogen and thrombin solutions must be carefully considered for cell delivery because they affect 3D fibrin clot structure and cell proliferation.