Bioactive chitosan biguanidine-based injectable hydrogels as a novel BMP-2 and VEGF carrier for osteogenesis of dental pulp stem cells.

Nowadays, vascularization and mineralization of bone defects is the main bottleneck in the bone regeneration field that is needed to be overcome and developed. Here, we prepared novel in-situ formed injectable hydrogels based on chitosan biguanidine and carboxymethylcellulose loaded with vascular endothelial growth factor (VEGF) and recombinant Bone morphogenetic protein 2 (BMP-2) and studied its influence on osteoblastic differentiation of dental pulp stem cells (DPSCs). The sequential release behavior of the VEGF and BMP-2 from hydrogels adjusted with the pattern of normal human bone growth. MTT assay exhibited that these hydrogels were non-toxic and significantly increased DPSCs proliferation. The Real-time PCR and Western blot analysis on CG11/BMP2-VEGF showed significantly higher gene and protein expression of ALP, COL1α1, and OCN. These results were confirmed by mineralization assay by Alizarin Red staining and Alkaline phosphatase enzyme activity. Based on these evaluations, these hydrogel holds potential as an injectable bone tissue engineering platform.

Onlay-graft of 3D printed Kagome-structure PCL scaffold incorporated with rhBMP-2 based on hyaluronic acid hydrogel.

The onlay-graft, one of the most difficult graft conditions, is used for diverse clinical conditions, including plastic and dental surgery. The graft should withstand continuous pressure from overlying tissues and have excellent bone formation capability in a limited bone contact situation. We recently developed a 3D printed Kagome-structured polycaprolactone (PCL) scaffold that has a stronger mechanical property. This study evaluated the clinical feasibility of this scaffold for onlay-graft use. The value of the scaffold containing recombinant human bone morphogenetic protein-2 in a hyaluronate-based hydrogel (rhBMP-2/HA) to enhance bone regeneration was also assessed. 3D-printed Kagome-PCL scaffolds alone (n= 12, group I) or loaded with rhBMP-2/HA (n= 12, group II) were grafted using a rat calvarial onlay-graft model. Following sacrifice at 2, 4, and 8 weeks, all 3D-printed Kagome-PCL scaffolds were accurately positioned and firmly integrated to the recipient bone. Micro-computed tomography and histology analyses revealed a constant height of the scaffolds over time in all animals. New bone grew into the scaffolds in both groups, but with greater volume in group II. These results suggest the promising clinical feasibility of the 3D-printed Kagome-PCL scaffold for onlay-graft use and it could substitute the conventional onlay-graft in the plastic and dental reconstructive surgery in the near future.

Latent TGF-ß Activation Is a Hallmark of the Tenascin Family.

Transforming growth factor-ß (TGF-ß) isoforms are secreted as inactive complexes formed through non-covalent interactions between bioactive TGF-ß entities and their N-terminal pro-domains called latency-associated peptides (LAP). Extracellular activation of latent TGF-ß within this complex is a crucial step in the regulation of TGF-ß activity for tissue homeostasis and immune cell function. We previously showed that the matrix glycoprotein Tenascin-X (TN-X) interacted with the small latent TGF-ß complex and triggered the activation of the latent cytokine into a bioactive TGF-ß. This activation most likely occurs through a conformational change within the latent TGF-ß complex and requires the C-terminal fibrinogen-like (FBG) domain of the glycoprotein. As the FBG-like domain is highly conserved among the Tenascin family members, we hypothesized that Tenascin-C (TN-C), Tenascin-R (TN-R) and Tenascin-W (TN-W) might share with TN-X the ability to regulate TGF-ß bioavailability through their C-terminal domain. Here, we demonstrate that purified recombinant full-length Tenascins associate with the small latent TGF-ß complex through their FBG-like domains. This association promotes activation of the latent cytokine and subsequent TGF-ß cell responses in mammary epithelial cells, such as cytostasis and epithelial-to-mesenchymal transition (EMT). Considering the pleiotropic role of TGF-ß in numerous physiological and pathological contexts, our data indicate a novel common function for the Tenascin family in the regulation of tissue homeostasis under healthy and pathological conditions.

Bone morphogenetic protein 2-induced cellular chemotaxis drives tissue patterning during critical-sized bone defect healing: an in silico study.

Critical-sized bone defects are critical healing conditions that, if left untreated, often lead to non-unions. To reduce the risk, critical-sized bone defects are often treated with recombinant human BMP-2. Although enhanced bone tissue formation is observed when BMP-2 is administered locally to the defect, spatial and temporal distribution of callus tissue often differs from that found during regular bone healing or in defects treated differently. How this altered tissue patterning due to BMP-2 treatment is linked to mechano-biological principles at the cellular scale remains largely unknown. In this study, the mechano-biological regulation of BMP-2-treated critical-sized bone defect healing was investigated using a multiphysics multiscale in silico approach. Finite element and agent-based modeling techniques were combined to simulate healing within a critical-sized bone defect (5 mm) in a rat femur. Computer model predictions were compared to in vivo microCT data outcome of bone tissue patterning at 2, 4, and 6 weeks postoperation. In vivo, BMP-2 treatment led to complete healing through periosteal bone bridging already after 2 weeks postoperation. Computer model simulations showed that the BMP-2 specific tissue patterning can be explained by the migration of mesenchymal stromal cells to regions with a specific concentration of BMP-2 (chemotaxis). This study shows how computational modeling can help us to further understand the mechanisms behind treatment effects on compromised healing conditions as well as to optimize future treatment strategies.

Structural basis for transcriptional coactivator recognition by SMAD2 in TGF-ß signaling.

Transforming growth factor-ß (TGF-ß) proteins regulate multiple cellular functions, including cell proliferation, apoptosis, and extracellular matrix formation. The dysregulation of TGF-ß signaling causes diseases such as cancer and fibrosis, and therefore, understanding the biochemical basis of TGF-ß signal transduction is important for elucidating pathogenic mechanisms in these diseases. SMAD proteins are transcription factors that mediate TGF-ß signaling-dependent gene expression. The transcriptional coactivator CBP directly interacts with the MH2 domains of SMAD2 to activate SMAD complex-dependent gene expression. Here, we report the structural basis for CBP recognition by SMAD2. The crystal structures of the SMAD2 MH2 domain in complex with the SMAD2-binding region of CBP showed that CBP forms an amphiphilic helix on the hydrophobic surface of SMAD2. The expression of a mutated CBP peptide that showed increased SMAD2 binding repressed SMAD2-dependent gene expression in response to TGF-ß signaling in cultured cells. Disrupting the interaction between SMAD2 and CBP may therefore be a promising strategy for suppressing SMAD-dependent gene expression.

Biomimetic UHMWPE/HA scaffolds with rhBMP-2 and erythropoietin for reconstructive surgery.

A promising direction for the replacement of expanded bone defects is the development of bioimplants based on synthetic biocompatible materials impregnated with growth factors that stimulate bone remodeling. Novel biomimetic highly porous ultra-high molecular weight polyethylene (UHMWPE)/40% hydroxyapatite (HA) scaffold for reconstructive surgery with the porosity of 85 ± 1% vol. and a diameter of pores in the range of 50-800 µm was developed. The manufacturing process allowed the formation of trabecular-like architecture without additional solvents and thermo-oxidative degradation. Biomimetic UHMWPE/HA scaffold was biocompatible and provided effective tissue ingrowth on a model of critical-sized cranial defects in mice. The combined use of UHMWPE/HA with Bone Morphogenetic Protein-2 (BMP-2) demonstrated intensive mineralized bone formation as early as 3 weeks after surgery. The addition of erythropoietin (EPO) significantly enhanced angiogenesis in newly formed tissues. The effect of EPO of bacterial origin on bone tissue defect healing was demonstrated for the first time. The developed biomimetic highly porous UHMWPE/HA scaffold can be used separately or in combination with rhBMP-2 and EPO for reconstructive surgery to solve the problems associated with difference between implant architecture and trabecular bone, low osteointegration and bioinertness.

Effect of osmolytes on in-vitro aggregation properties of peptides derived from TGFBIp.

Protein aggregation has been one of the leading triggers of various disease conditions, such as Alzheimer's, Parkinson's and other amyloidosis. TGFBI-associated corneal dystrophies are protein aggregation disorders in which the mutant TGFBIp aggregates and accumulates in the cornea, leading to a reduction in visual acuity and blindness in severe cases. Currently, the only therapy available is invasive and there is a known recurrence after surgery. In this study, we tested the inhibitory and amyloid dissociation properties of four osmolytes in an in-vitro TGFBI peptide aggregation model. The 23-amino acid long peptide (TGFBIp 611-633 with the mutation c.623 G>R) from the 4th FAS-1 domain of TGFBIp that rapidly forms amyloid fibrils was used in the study. Several biophysical methods like Thioflavin T (ThT) fluorescence, Circular Dichroism (CD), fluorescence microscopy and Transmission electron microscopy (TEM) were used to study the inhibitory and amyloid disaggregation properties of the four osmolytes (Betaine, Raffinose, Sarcosine, and Taurine). The osmolytes were effective in both inhibiting and disaggregating the amyloid fibrils derived from TGFBIp 611-633 c.623 G>R peptide. The osmolytes did not have an adverse toxic effect on cultured human corneal fibroblast cells and could potentially be a useful therapeutic strategy for patients with TGFBIp corneal dystrophies.

TGFBR2 mediated phosphorylation of BUB1 at Ser-318 is required for transforming growth factor-ß signaling.

BUB1 (budding uninhibited by benzimidazoles-1) is required for efficient TGF-ß signaling, through its role in stabilizing the TGFBR1 and TGFBR2 complex. Here we demonstrate that TGFBR2 phosphorylates BUB1 at Serine-318, which is conserved in primates. S318 phosphorylation abrogates the interaction of BUB1 with TGFBR1 and SMAD2. Using BUB1 truncation domains (1-241, 241-482 and 482-723), we demonstrate that multiple contact points exist between BUB1 and TGF-ß signaling components and that these interactions are independent of the BUB1 tetratricopeptide repeat (TPR) domain. Moreover, substitutions in the middle domain (241-482) encompassing S318 reveals that efficient interaction with TGFBR2 occurs only in its dephosphorylated state (241-482 S318A). In contrast, the phospho-mimicking mutant (241-482 S318D) exhibits efficient binding with SMAD2 and its over-expression results in a decrease in TGFBR1-TGFBR2 and TGFBR1-SMAD2 interactions. These findings suggest that TGFBR2 mediated BUB1 phosphorylation at S318 may serve as a switch for the dissociation of the SMAD2-TGFBR complex, and therefore represents a regulatory event for TGF-ß signaling. Finally, we provide evidence that the BUB1-TGF-ß signaling axis may mediate aggressive phenotypes in a variety of cancers.

Nanoclay-functionalized 3D nanofibrous scaffolds promote bone regeneration.

Developing a biomaterial that can promote osteoblastic differentiation, thereby reducing the needs of exogenous osteogenic factors for large bone repair, has been a significant and long-term technical hurdle. In this study, we developed an innovative nanoclay (nanosilicate, NS)-functionalized 3D gelatin nanofibrous scaffold (GF/NS) through a thermally induced phase separation method together with the particle leaching technique (TIPS&P). In addition to the significantly higher mechanical strength, the composite scaffolds (GF/NS) demonstrated a significantly stronger ability to promote the osteogenic differentiation of human mesenchymal stem cells (hMSCs) in vitro compared to the GF scaffold. Our data further revealed that this intriguing pro-osteoblastic functionality was largely because of the unique features of NS, particularly, the strong binding ability to pro-osteoblastic factors (e.g., BMP2) as well as the intrinsic osteoinductivity of its bioactive degradation products. Most importantly, our in vivo studies indicated that GF/NS scaffolds significantly improved low-dose BMP2-induced ectopic bone regeneration in mice.

Thinking beyond tradition: Polyphenols as effective refolding modulators.

Despite polyphenols having had proven roles as amyloid alleviators their service has rarely been made use of in protein refolding/renaturation thus far, where aggregation can be a major competing pathway. TGFß3, expressed in inclusion bodies, is a classical example of a protein prone to high rate of aggregation severely limiting its refolding yield owing to its large cysteine content and structural complexity. Here, we have used various polyphenols (EGCG, baicalein, myricetin) either alone or in combination with the pseudo-chaperone beta cyclodextrin, in the refolding buffer. With the help of non-reducing SDS PAGE and size exclusion chromatography, we showed that refolding in the presence of baicalein or EGCG along with ßCD indeed increase the yield of the native protein in a time dependent manner. EGCG expedites the refolding process giving a maximum increase of the refolding yield within 24 h while baicalein takes as long as 48 h for the same. The mechanism of mode of actions of polyphenols during refolding was further delineated by ITC. The effect of polyphenols on the aggregation kinetics and stability of native TGFß3 were also explored. Thus these small molecules provide a promising alternate route in increasing the yield of aggregation prone proteins during refolding.

Improved accumulation of TGF-ß by photopolymerized chitosan/silk protein bio-hydrogel matrix to improve differentiations of mesenchymal stem cells in articular cartilage tissue regeneration.

Articular cartilage regeneration is a challenging process due to its inadequate ability of self-recovering biological mechanisms. The progresses of cartilage tissue engineering is supported to overwhelmed the repairing difficulties and degenerative diseases. The main goal of the present study is to design biomaterials with suitable physico-chemical, mechanical and biological properties for the carrier of growth factor and improving differentiation of mesenchymal stem cell into damaged cartilage tissues. Herein, TGF-ß loaded hydrogel network was prepared through the chemical interactions between vinyl group of natural polymers. Fourier-transform infrared spectroscopy results show the characteristic peaks at 3074 cm-1, 1713 cm-1, and 810 cm-1, which confirm the existence of the vinyl group and successful formation of maleoyl functionalized Chitosan (MCh). The obtained MCh was freely dissolved in the distilled water up to 8% (w/v). X-ray photoelectron spectroscopy survey spectral results show a peak at 289.0 eV which revealed that the OCO and DS were 1.2% and also evidenced the methacryl substitution of Silk fibroin (SF) nanoformulations. The weight loss and mechanical test were analyzed and the results showed that MSF acts as a foremost crosslinking point with MCh through the reaction between the methacrylate groups of MSF and maleoyl groups of MCh which led to enhancing the density and improved the compressive strength. The maximum drug release activity was recorded in the TGF-ß loaded MCh@MSF hydrogel compared to bare MCh hydrogel. Further, the TGF-ß loaded MCh@ MSF hydrogel exhibited the cell viability percentage nearly at 79-102% for MC3T3-E1 and 88-104% for BMDSCs. Similarly, the TGF-ß loaded MCh@MSF exhibited the highest inhibitory activity against E. coli (83%) than S. aureus (67%). Overall, this study concluded the TGF-ß loaded MCh@MSF showed better biocompatibility and could be utilized in the field of cartilage tissue engineering.

Multifunctional fibrous scaffolds for bone regeneration with enhanced vascularization.

Due to the structural similarity to the extracellular matrix of human tissue and the ultra-high surface area-to-volume ratio, three dimensional electrospun fibrous structures have been increasingly used as tissue engineering scaffolds. Given that successful bone regeneration requires both good osteogenesis and vascularization, producing scaffolds that have both osteogenic and angiogenic potential is highly desirable. In this investigation, tricomponent fibrous scaffolds simultaneously incorporated with recombinant human vein endothelial growth factor (rhVEGF), recombinant human bone morphogenetic protein-2 (rhBMP-2) and bioactive calcium phosphate (Ca-P) nanoparticles are produced through a novel multi-source multi-power electrospinning method, and sequential growth factor release with a quick rhVEGF release and a steady rhBMP-2 release is achieved. The enhanced human umbilical vein endothelial cell (HUVEC) migration and tube formation, and up-regulated human bone marrow derived mesenchymal stem cell (hBMSC) osteogenic differentiation and mineralization demonstrate that tricomponent scaffolds have balanced angiogenic-osteogenic properties in vitro. 8 weeks after the scaffold implantation into the cranial defects of mice, obvious new bone regeneration and newly formed capillaries are observed in tricomponent scaffolds, suggesting that the tricomponent scaffolds enhance osteogenesis in vivo with required vascularization, which shows the great potential of the tricomponent scaffolds in bone tissue regeneration.

Soluble expression and purification of high-bioactivity recombinant human bone morphogenetic protein-2 by codon optimisation in Escherichia coli.

We developed a simple method of preparing recombinant human bone morphogenetic protein-2 (rhBMP-2) with high biological activity. This rhBMP-2 was overproduced in Escherichia coli as a fusion protein with thioredoxin 6xHis-tag at its amino terminus. The cDNA fragment of human bone morphogenetic protein-2 (hBMP-2) fused to the secretion signal of alkaline phosphatase (PhoA) was expressed under T7 promoter in E. coli. After DNA sequence confirmation, the recombinant vector pETpho-bmp2 was transformed into E. coli BL21 (DE3). rhBMP-2 was produced by the recombinant strain pETpho-bmp2/BL21 (DE3) in a soluble form with an yield of 6.2 mg/L culture. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) results showed that the molecular weight of the product was approximately 28 kD. Moreover, rhBMP-2 was secreted as a dimer with a natural structure. rhBMP-2, purified by Ni Nitrilotriacetic acid Agarose (Ni-NTA) affinity chromatography, was used to examine osteosarcoma MG-63 cells and assay the alkaline phosphatase (ALP) activity. Results showed that rhBMP-2 induced MG-63 cell differentiation. When the final concentration was 500 ng/mL, the effect was more remarkable and ALP activity reached 525% compared with that of the control group.

Structural consequences of transforming growth factor beta-1 activation from near-therapeutic X-ray doses.

Dissociation of transforming growth factor beta-1 (TGFß-1) from the inhibitory protein latency-associated peptide (LAP) can occur from low doses of X-ray irradiation of the LAP-TGFß-1 complex, resulting in the activation of TGFß-1, and can have health-related consequences. Using the tools and knowledge developed in the study of radiation damage in the crystallographic setting, small-angle X-ray scattering (SAXS) and complementary techniques suggest an activation process that is initiated but not driven by the initial X-ray exposure. LAP is revealed to be extended when not bound to TGFß-1 and has a different structural conformation compared to the bound state. These studies pave the way for the structural understanding of systems impacted at therapeutic X-ray doses and show the potential impact of radiation damage studies beyond their original intent.

Carbodiimide Conjugation of Latent Transforming Growth Factor ß1 to Superparamagnetic Iron Oxide Nanoparticles for Remote Activation.

Conjugation of latent growth factors to superparamagnetic iron oxide nanoparticles (SPIONs) is potentially useful for magnetically triggered release of bioactive macromolecules. Thus, the goal of this work was to trigger the release of active Transforming Growth-Factor Beta (TGF-ß) via magnetic hyperthermia by binding SPIONs to the latent form of TGF-ß, since heat has been shown to induce release of TGF-ß from the latent complex. Commercially available SPIONS with high specific absorption rates (SAR) were hydrolyzed in 70% ethanol to create surface carboxylic acid conjugation sites for carbodiimide chemistry. Fourier-Transform Infra-Red (FTIR) analysis verified the conversion of maleic anhydride to maleic acid. 1-Ethyl-2-(3-dimethyulaminopropyl) carbodiimide (EDC) and N-hydroxysulfosuccinimide (Sulfo-NHS) were used to bind to the open conjugation sites of the SPION in order to graft latent TGF-ß onto the particles. The resulting conjugated particles were imaged with transmission electron microscopy (TEM), and the complexed particles were characterized by dynamic light scattering (DLS) and superconducting quantum interference device (SQUID) magnetometry. Enzyme-linked immunosorbent assay (ELISA) was used to assess the thermally triggered release of active TGF-ß from the latent complex, demonstrating that conjugation did not interfere with release. Results showed that latent TGF-ß was successfully conjugated to the iron oxide nanoparticles, and magnetically triggered release of active TGF-ß was achieved.

The serine protease HtrA1 cleaves misfolded transforming growth factor ß-induced protein (TGFBIp) and induces amyloid formation.

The serine protease high-temperature requirement protein A1 (HtrA1) is associated with protein-misfolding disorders such as Alzheimer's disease and transforming growth factor ß-induced protein (TGFBIp)-linked corneal dystrophy. In this study, using several biochemical and biophysical approaches, including recombinant protein expression, LC-MS/MS and 2DE analyses, and thioflavin T (ThT) fluorescence assays for amyloid fibril detection, and FTIR assays, we investigated the role of HtrA1 both in normal TGFBIp turnover and in corneal amyloid formation. We show that HtrA1 can cleave WT TGFBIp but prefers amyloidogenic variants. Corneal TGFBIp is extensively processed in healthy people, resulting in C-terminal degradation products spanning the FAS1-4 domain of TGFBIp. We show here that HtrA1 cleaves the WT FAS1-4 domain only inefficiently, whereas the amyloidogenic FAS1-4 mutations transform this domain into a considerably better HTRA1 substrate. Moreover, HtrA1 cleavage of the mutant FAS1-4 domains generated peptides capable of forming in vitro amyloid aggregates. Significantly, these peptides have been previously identified in amyloid deposits in vivo, supporting the idea that HtrA1 is a causative agent for TGFBIp-associated amyloidosis in corneal dystrophy. In summary, our results indicate that TGFBIp is an HtrA1 substrate and that some mutations in the gene encoding TGFBIp cause aberrant HtrA1-mediated processing that results in amyloidogenesis in corneal dystrophies.

Lysostaphin and BMP-2 co-delivery reduces S. aureus infection and regenerates critical-sized segmental bone defects.

Staphylococcus aureus is the most common pathogen associated with bacterial infections in orthopedic procedures. Infections often lead to implant failure and subsequent removal, motivating the development of bifunctional materials that both promote repair and prevent failure due to infection. Lysostaphin is an anti-staphylococcal enzyme resulting in bacterial lysis and biofilm reduction. Lysostaphin use is limited by the lack of effective delivery methods to provide sustained, high doses of enzyme to infection sites. We engineered a BMP-2-loaded lysostaphin-delivering hydrogel that simultaneously prevents S. aureus infection and repairs nonhealing segmental bone defects in the murine radius. Lysostaphin-delivering hydrogels eradicated S. aureus infection and resulted in mechanically competent bone. Cytokine and immune cell profiling demonstrated that lysostaphin-delivering hydrogels restored the local inflammatory environment to that of a sterile injury. These results show that BMP-2-loaded lysostaphin-delivering hydrogel therapy effectively eliminates S. aureus infection while simultaneously regenerating functional bone resulting in defect healing.

Preparation of beta-tricalcium phosphate microsphere-hyaluronic acid-based powder gel composite as a carrier for rhBMP-2 injection and evaluation using long bone segmental defect model.

The specific objective of this study was to evaluate whether rhBMP-2-loaded bio-scaffolds can be used as effective rhBMP-2 carriers in the implantation of bone defect sites or poor bone quality in host bone. The rhBMP-2 release pattern test showed slow results in both groups, and a 1:9 ratio composition with a high water-absorption rate was selected for in vivo study. All animals euthanized after 9 weeks. The new bone formation and bone quantity and quality of fibular samples were examined. The results showed that the rhBMP-2 powder gel composite improved the new bone formation in the cortical bone and the marrow space. The length of new bone formation ratio of the rhBMP-2 loaded composite group was significantly higher than the powder gel group. The composite of powder gel seems to be a nice carrier, and slow release of rhBMP-2 can promote new bone formation in a segmental cortical bone defect after implantation.