Cholesteryl group- and acryloyl group-bearing pullulan nanogel to deliver BMP2 and FGF18 for bone tissue engineering

To create a drug delivery system that allows the controlled release of proteins, such as growth factors, over a long-term period, cholesteryl group- and acryloyl group-bearing pullulan (CHPOA) nanogels were aggregated to form fast-degradable hydrogels (CHPOA/hydrogels) by cross-linking with thiol-bearing polyethylene glycol. The gold standard of clinical bone reconstruction therapy with a physiologically active material is treatment with recombinant human bone morphogenetic protein 2 (BMP2); however, this approach has limitations, such as inflammation, poor cost-efficiency, and varying interindividual susceptibility. In this study, two distinct growth factors, BMP2 and recombinant human fibroblast growth factor 18 (FGF18), were applied to a critical-size skull bone defect for bone repair by the CHPOA/hydrogel system. The CHPOA-FGF18/hydrogel displayed identical results to the control CHPOA-PBS/hydrogel, and the CHPOA-BMP2/hydrogel treatment imperfectly induced bone repair. By contrast, the CHPOA-FGF18?+?BMP2/hydrogel treatment strongly enhanced and stabilized the BMP2-dependent bone repair, inducing osteoprogenitor cell infiltration inside and around the hydrogel. This report indicates that the CHPOA/hydrogel system can successfully deliver two different proteins to the bone defect to induce effective bone repair. The combination of the CHPOA/hydrogel system with the growth factors FGF18 and BMP2 might be a step towards efficient bone tissue engineering.     

Effects of Adipose Tissue Stem Cell Concurrent with Greater Omentum on Experimental Long-Bone Healing in Dog

Repair of large bone defects resulting from trauma, tumors, and osteitis is a current challenge to surgeons. Adipose-derived adult stem cells (ASCs) are multipotent cells that are able to differentiate into osteoblasts in the presence of certain factors. In this study, the role of greater omentum as a scaffold incorporation of ASCs was evaluated in long-bone defect healing in dog model. Sixteen 3–4-year-old, male adult mongrel dogs, weighing 25.2 ± 3.5 kg, were used in this study. In the control group (n = 4), the defect was left empty. In the omental group (n = 4), the defect was filled with harvested omentum. In the omental-ASCs group (n = 4), the defect was filled with omentum and 1 mL of ASCs was injected into the grafted omentum. In the omental-culture medium group (n = 4), 1 mL of culture medium was injected into the grafted omentum. Finally, the injured radial bones were fixed with plate and screw. Radiographs of each forelimb was taken postoperatively on the first day and at the second, fourth, sixth, and eighth weeks postinjury to evaluate bone formation, union, and remodeling of the defect. The operated radii were removed on the 56th postoperative day and were histopathologically evaluated. In this study, both omental-culture medium and omental-ASCs groups demonstrated superior osteogenic potential in healing the radial bone defect. Compared to those of the omental and control groups, more advanced bone healing criteria were present in the omental-culture medium and omental-ASCs groups at radiological and histopathological levels at 8 weeks postsurgery.     

Bone regeneration using an alpha 2 beta 1 integrin-specific hydrogel as a BMP-2 delivery vehicle

Non-healing bone defects present tremendous socioeconomic costs. Although successful in some clinical settings, bone morphogenetic protein (BMP) therapies require supraphysiological dose delivery for bone repair, raising treatment costs and risks of complications. We engineered a protease-degradable poly(ethylene glycol) (PEG) synthetic hydrogel functionalized with a triple helical, α2β1 integrin-specific peptide (GFOGER) as a BMP-2 delivery vehicle. GFOGER-functionalized hydrogels lacking BMP-2 directed human stem cell differentiation and produced significant enhancements in bone repair within a critical-sized bone defect compared to RGD hydrogels or empty defects. GFOGER functionalization was crucial to the BMP-2-dependent healing response. Importantly, these engineered hydrogels outperformed the current clinical carrier in repairing non-healing bone defects at low BMP-2 doses. GFOGER hydrogels provided sustained in vivo release of encapsulated BMP-2, increased osteoprogenitor localization in the defect site, enhanced bone formation and induced defect bridging and mechanically robust healing at low BMP-2 doses which stimulated almost no bone regeneration when delivered from collagen sponges. These findings demonstrate that GFOGER hydrogels promote bone regeneration in challenging defects with low delivered BMP-2 doses and represent an effective delivery vehicle for protein therapeutics with translational potential.     

Microfluidic assay of endothelial cell migration in 3D interpenetrating polymer semi-network HA-Collagen hydrogel

Cell migration through the extracellular matrix (ECM) is one of the key features for physiological and pathological processes such as angiogenesis, cancer metastasis, and wound healing. In particular, the quantitative assay of endothelial cell migration under the well-defined three dimensional (3D) microenvironment is important to analyze the angiogenesis mechanism. In this study, we report a microfluidic assay of endothelial cell sprouting and migration into an interpenetrating polymer semi-network HA-Collagen (SIPNs CH) hydrogel as ECM providing an enhanced in vivo mimicking 3D microenvironment to cells. The microfluidic chip could provide a well-controlled gradient of growth factor to cells, whereas the hydrogel could mimic a well-defined 3D microenvironment in vivo. (In addition/Furthermore, the microfluidic chip gives a well-controlled gradient of growth factor to cells) For this reason, three types of hydrogel, composed of semi-interpenetrating networks of collagen and hyaluronic acid were prepared, and firstly we proved the role of the hydrogel in endothelial cell migration. The diffusion property and swelling ratio of the hydrogel were characterized. It modulated the migration of endothelial cells in quantified manner, also being influenced by additional synthesis of Matrix metalloproteinase(MMP)-sensitive remodeling peptides and Arginine–glycine–lycinee (RGD) cell adhesion peptides. We successfully established a novel cell migration platform by changing major determinants such as ECM material under biochemical synthesis and under growth factor gradients in a microfluidic manner.     

Spontaneous Healing Capacity of Calvarial Bone Defects in mdx Mice

The mdx mouse is an experimental model widely used for the study of Duchenne muscular dystrophy, which is characterized by the lack of dystrophin and cycles of muscle degeneration/regeneration. Studies demonstrated elevated levels of growth factors and accelerated skin wound repair in these animals. We therefore raised the hypothesis that the bone repair process might also be altered in these animals. Thus, the objective of this study was to evaluate the spontaneous healing of calvarial defects in mdx mice by histomorphometric analysis. Animals (45 days old) were divided into mdx and control groups. A defect measuring 2 mm in diameter was produced surgically in the right parietal bone of each animal. The animals were sacrificed 15, 30, and 60 days after surgery, and the skulls were processed by routine histological procedures. No difference in the volume of new bone inside the defect was observed between the two groups at any of the three postoperative time points. There was also no difference between the different periods of healing when each group was analyzed separately. The lower quality of femoral and calvarial bone in mdx mice reported in previous studies and the similar bone regeneration rates seen in two groups suggest that the healing capacity of calvarial defects was more expressive in mdx mice than in control animals. An increase in the amount of osteogenic factors released by damaged myofibers may have favored osteogenesis during bone defect healing in mdx mice. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.     

Modulation of the Keratinocyte–Fibroblast Paracrine Relationship with Gelatin-Based Semi-interpenetrating Networks Containing Bioactive Factors for Wound Repair

Gelatin-based semi-interpenetrating networks (sIPNs) containing soluble and covalently-linked bioactive factors have been shown to aid in wound healing; however, the biological responses elicited by the introduction of sIPN biomaterials remain unclear. In the current study, modulation of the re-epithelialization phase of wound healing by sIPNs grafted with PEGylated fibronectin-derived peptides and utilized as platforms for the delivery of exogenous keratinocyte growth factor (KGF) was evaluated. Following wounding, keratinocyte migration, proliferation and protein secretion is largely controlled by diffusible factors, such as KGF, released by the underlying fibroblasts. The impact of sIPNs and exogenous KGF upon the latter keratinocyte–fibroblast paracrine relationship and keratinocyte behavior was explored by monitoring keratinocyte adhesion and cytokine (IL-1α, IL-1β, IL-6, KGF, GM-CSF and TGF-α) release. Results were generally similar for keratinocyte monoculture and keratinocyte–fibroblast co-culture systems. Although keratinocyte adhesion increased over time for positive control surfaces, adhesion to the sIPNs remained low throughout the course of the study. Release of IL-1α and GM-CSF was increased by exogenous KGF. The effects were more noticeable on the positive control surfaces relative to the sIPN surfaces. Regulation of the release of TGF-α was surface dependent, while IL-6 release was dependent upon surface type, the inclusion of exogenous KGF and the presence of fibroblasts. The findings indicate that during re-epithelialization, sIPNs containing soluble bioactive factors aid in wound healing primarily by serving as conduits for KGF, which induces the release of other key cytokines involved in tissue repair.     

Bone healing of commercial oral implants with RGD immobilization through electrodeposited poly(ethylene glycol) in rabbit cancellous bone

Immobilization of RGD peptides on titanium (Ti) surfaces enhances implant bone healing by promoting early osteoblastic cell attachment and subsequent differentiation by facilitating integrin binding. Our previous studies have demonstrated the efficacy of RGD peptide immobilization on Ti surfaces through the electrodeposition of poly(ethylene glycol) (PEG) (RGD/PEG/Ti), which exhibited good chemical stability and bonding. The RGD/PEG/Ti surface promoted differentiation and mineralization of pre-osteoblasts. This study investigated the in vivo bone healing capacity of the RGD/PEG/Ti surface for biomedical application as a more osteoconductive implant surface in dentistry. The RGD/PEG/Ti surface was produced on an osteoconductive implant surface, i.e. the grit blasted micro-rough surface of a commercial oral implant. The osteoconductivity of the RGD/PEG/Ti surface was compared by histomorphometric evaluation with an RGD peptide-coated surface obtained by simple adsorption in rabbit cancellous bone after 2 and 4 weeks healing. The RGD/PEG/Ti implants displayed a high degree of direct bone apposition in cancellous bone and achieved greater active bone apposition, even in areas of poor surrounding bone. Significant increases in the bone to implant contact percentage were observed for RGD/PEG/Ti implants compared with RGD-coated Ti implants obtained by simple adsorption both after 2 and 4 weeks healing (P<0.05). These results demonstrate that RGD peptide immobilization on a Ti surface through electrodeposited PEG may be an effective method for enhancing bone healing with commercial micro-rough surface oral implants in cancellous bone by achieving rapid bone apposition on the implant surface.     

Development of anti-adhesive spongy sheet composed of hyaluronic acid and collagen containing epidermal growth factor

Anti-adhesive products need to be designed while considering the concept of wound healing. Two main events must proceed simultaneously: facilitating wound healing in surgically excised tissue, as well as preventing injured tissue from adhering to the surrounding tissue. The present study aimed to develop an anti-adhesive spongy sheet composed of hyaluronic acid and collagen (Col) containing epidermal growth factor, and to investigate the potential of this spongy sheet using an in vitro wound surface model (placing a spongy sheet on a fibroblast-incorporating Col gel sheet) and an in vitro inter-tissue model (placing a spongy sheet between two fibroblast-incorporating Col gel sheets). These in vitro experiments demonstrated that this spongy sheet effectively stimulates fibroblasts to release an increased amount of vascular endothelial growth factor and hepatocyte growth factor, which are essential for wound healing to proceed succesfully. In addition, anti-adhesive performance of this spongy sheet was evaluated in animal experiments using Sprague Dawley rats. Under anesthesia, a 1?cm?×?2?cm segment of peritoneum was superficially excised from walls, and the cecum was then abraded by scraping with a scalpel blade over a 1?cm?×?2?cm area. A piece of spongy sheet was placed on the peritoneal defect. Both defects were placed in contact, and the incision was closed by suturing. Peritoneal condition was evaluated after one week. This spongy sheet was capable of facilitating the wound healing of surgically excised tissue and preventing surgically excised tissue from adhering to surrounding tissues.     

Inhibition of tumor implantation at sites of trauma by Arg-Gly-Asp containing proteins and peptides

We report on the inhibition of wound implantation by TA3Ha mammary carcinoma cells by Arg-Gly-Asp containing proteins and peptides using a hepatic wedge resection model. Intravenously injected TA3Ha cells rarely form tumor in the liver of syngeneic mice, but after hepatic wedge resection, 45% (107/240) of the mice develop tumors in the hepatic wound. Hepatic wound implantation is significantly (P = 0.01) inhibited by pretreating the cells with whole mouse plasma, but not with fibrinogen-depleted plasma or serum. Tumor inhibition is also achieved by pretreatment of cells with fibrinogen (P = 0.05–0.0004), fibronectin (P = 0.007) and laminin, but not by albumin. The active domain appears to be the RGDS sequence since the deca- and tetrapeptides containing RGDS inhibit wound implantation (P < 0.05). However, the tetrapeptide Arg-Gly-Glu-Ser has no such activity. None of these agents affects ascites tumor formation by the intraperitoneally injected cells, suggesting that anchorage independent growth of cells is not affected. We propose that proteins and peptides containing RGD occupy the binding sites and prevent the cells from interacting with cell adhesion proteins in healing wounds. Proteins and/or peptides containing RGD may be useful for preventing local recurrence in postsurgical cancer patients.     

A Porous Hydroxyapatite/Gelatin Nanocomposite Scaffold for Bone Tissue Repair: In Vitro and In Vivo Evaluation

Abstract

In this study, a nano-structured scaffold was designed for bone repair using hydroxapatite and gelatin as its main components. The scaffold was prepared via layer solvent casting combined with freeze-drying and lamination techniques and characterized by the commonly used bulk techniques. The biocompatibility and osteoconductivity of this scaffold and its capacity to promote bone healing were also evaluated. Osteoblast-like cells were seeded on these scaffolds and their proliferation rate, intracellular alkaline phosphatase (ALP) activity and ability to form mineralized bone nodules were compared with those osteoblasts grown on cell culture plastic surfaces. Also, the scaffolds were implanted in a critical bone defect created on rat calvarium. Engineering analyses show that the scaffold posses a three dimensional interconnected homogenous porous structure with a porosity of about 82% and pore sizes ranging from 300 to 500 μm. Mechanical indices are in the range of spongy bones. The results obtained from biological assessment show that this scaffold does not negatively affect osteoblasts proliferation rate and improves osteoblasts function as shown by increasing the ALP activity and calcium deposition and formation of mineralized bone nodules. In addition, the scaffold promoted healing of critical size calvarial bone defect in rats. It could be concluded that this scaffold fulfills all the main requirements to be considered as a bone substitute.     

The degree of correction in open-wedge high tibial osteotomy compromises bone healing: A consecutive review of 101 cases

BackgroundThe bone healing in open-wedge high tibial osteotomy (OWHTO) proceeds gradually by a filling of the osteotomy gap. This can comprise several risk factors.MethodsA retrospective study analysed the clinical and radiological course of 101 consecutive OWHTOs in 96 patients. The following risk factors were considered: age, body mass index, tobacco consumption, amount of tobacco consumption, severity of comorbidities, infection of the surgical area, occurrence of a lateral hinge fracture and the degree of correction. The bone healing was evaluated by using the modified Radiographic Union Score for Tibial fractures (RUST).ResultsA disturbance in bone healing was observed in 16 of the 101 osteotomies. Binary logistic regression analysis showed a correlation between the angle of the opening wedge and the development of a disturbance in bone healing (P = 0.002). The odds ratio indicated an increase in the risk of a disturbance in bone healing of 56% with each additional degree of correction. For the risk factor ‘age’ a statistical trend was recognizable (P = 0.077) with the risk of a disturbance in bone healing in higher age.ConclusionLateral hinge fractures seem not to have a detrimental effect on the filling of the osteotomy gap. An increase in the opening wedge bears the risk of a disturbance in bone healing.     

Wound pH-Responsive Sustained Release of Therapeutics from a Poly(NIPAAm-co-AAc) Hydrogel

Wound pH strongly influences residence time and activity of various growth factors during wound healing. Hence, a pH-responsive sustained release growth factor delivery system could be beneficial for effective treatment of wound. In this context, an effort was made to evaluate the potential of a poly(N-isopropylacrylamide-co-acrylic acid) hydrogel as pH-sensitive sustained release system for wound-pH-dependent therapeutics delivery. The polymer was synthesized via radical copolymerization and influence of pH on lower critical solution temperature (LCST), microarchitechture and swelling of the hydrogel was evaluated. Results showed a pH-dependent variation in the physical properties of the hydrogel within the wound pH range. Fluorescence recovery after photobleaching (FRAP) analysis endorsed a pH dependent restricted diffusion of the BSA in the hydrogel. Later, release of bovine serum albumin (BSA), vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) (each 5%, w/v) from the hydrogel within the range of wound pH (pH 6.7–7.9) were examined. Analysis showed non-Fickian release of therapeutics from the hydrogel with a significant variation in release rate and cumulative release with the increase in pH. Retention of the bioactivity of the released EGF was confirmed by studying murine dermal fibroblast cell proliferation in vitro. Finally, a growth factor (EGF or VEGF)-loaded hydrogel was applied on a murine excisional wound model and showed augmentation of wound healing in comparison to conventional sustained release growth factor therapy.     

Augmented bone regeneration activity of platelet-rich plasma by biodegradable gelatin hydrogel

This study investigates the ability of platelet-rich plasma (PRP) combined with biomaterials to enhance in vivo bone-repairing activity. A biodegradable hydrogel was prepared from gelatin, which has an affinity for various growth factors. Rabbit PRP was conventionally prepared by blood centrifugation and dropped onto freeze-dried gelatin hydrogel to obtain gelatin hydrogel incorporating PRP. Gelatin hydrogel incorporating PRP was applied to a bone defect of rabbit ulna to evaluate bone formation at the defect in terms of soft X-ray and histological examinations. As controls, fibrin incorporating PRP, empty gelatin hydrogel, and free PRP were applied to the defect; in addition, defect without any application was examined. Successful bone regeneration was observed at bone defect treated with gelatin hydrogel incorporating PRP, in marked contrast to the control groups. When in contact with gelatin, growth factors, such as platelet-derived growth factor and transforming growth factor beta(1), were released from the PRP. PRP growth factors are immobilized in the hydrogel through physicochemical interaction with gelatin molecules. The immobilized growth factors are released from the hydrogel in concert with hydrogel degradation. It is likely that the gelatin hydrogel permitted the controlled release of bioactive growth factors, resulting in factor-induced promotion of bone regeneration.     

Interaction with the stroma and endothelium

Prostatic carcinoma cells have a propensity to metastasize to bone, and we propose that this phenomenon may be promoted by the adhesion of metastatic cells to bone matrix. Bone matrix is produced by osteoblasts, and we have developed an in vitro model of bone matrix by isolating the substratum deposited by human osteoblast-like U20S cells. The collagenous nature of this matrix was demonstrated by the incorporation of [³H]proline and its subsequent release by purified collagenase. Both U20S matrix and purified type I collagen stimulated the adhesion of human PC-3 prostatic carcinoma cells. Human laminin supported adhesion to a much lesser extent, and PC-3 cells did not adhere to fibronectin. Adhesion of PC-3 cells to U20S matrix closely resembled adhesion to purified type I collagen with respect to (a) inhibition by a collagen-derived peptide and by antibodies raised against α2 or β1 integrin collagen receptor subunits; (b) lack of inhibition by RGD (Arg-Gly-Asp) peptides; (c) stimulation by Mn²⁺ and Mg²⁺ ions but not by Ca²⁺ ion; and (d) stimulation by the phorbol ester PMA (phorbol 12-myristate 13-acetate). This adhesion was also stimulated (2.3-fold) by transforming growth factor β (TGF-β), which is a major bone-derived growth factor. We conclude that human osteoblast-like matrix is an adhesive substrate for PC-3 prostate carcinoma cells. This adhesion appears to be mediated by the interaction of α2β1 integrins on PC-3 cells with matrix-derived collagen. The stimulation of this adhesion by TGF-β suggests that the co-expression of TGF-β and type I collagen in bone may synergistically facilitate the adhesion of metastatic cells to bone matrix proteins and thereby increase their localization in the skeleton.     

Sustained Release of Antimicrobial Peptide from Self-Assembling Hydrogel Enhanced Osteogenesis

Abstract

Biomaterials have been widely used in bone infection and osteomyelitis resulting from their versatile functionalities. As far as we know, the appearance of osteomyelitis was mainly caused by bacteria. Therefore, a biomaterial that can cure bone infection and promote osteogenesis may become an ideal candidate for the treatment of osteomyelitis. Cationic antimicrobial peptides (AMPs) have been proved to have an excellent ability to kill bacteria, fungi, viruses, and parasites. However, the application of AMPs in bone infection and osteomyelitis is quite limited. Here, we designed a new hydrogel that has an inhibitory effect on the proliferation of S. aureus and enhances osteogenesis. RADA16 self-assembling peptide has been applied for AMPs delivery. In this study, we demonstrated that RADA16 could form a stable structure and afford the sustained release of AMPs. The interwoven nanofiber morphology was detected by field emission scanning electron microscopy. The sustained release study revealed that the release of AMPs could be obtained until 28 days. In vitro research showed this new self-assembling hydrogel could promote the proliferation of bone mesenchymal stem cells (BMSCs) and inhibited the growth of S. aureus. More importantly, the results in vivo also proved that RADA16-AMP self-assembling peptide had an excellent effect on bone formation. Our findings implied that we successfully combined RADA16 and AMPs together and laid the foundation for the application of this new hydrogel and open new avenues for biomaterials.     

Effectiveness of topical application of ostrich oil on the healing of Staphylococcus aureus- and Pseudomonas aeruginosa-infected wounds

Background/aims: Management of infected wounds is one of the major challenges that surgeons and nurses face. Several antimicrobial agents have been used, but the toxicity, drug resistance, and their effect on the healing process remain a matter of concern. The present study was designed to analyze the accelerative impact of topical application of ostrich oil on infected wounds in a mouse model. Materials and methods: 72 BALB/c mice were divided into four main groups of control-sham, mupirocin, and two treatment groups receiving 2% and 4% (w/w) concentrations of ostrich oil, topically. The mice were routinely anesthetized and wounds were created by excising the skin with a 5-mm biopsy punch. Immediately after wounding, an aliquot of 25 × 10⁷ Staphylococcus aureus and Pseudomonas aeruginosa was suspended in 50-μL phosphate-buffered saline and applied on the wound and the wound was left open. The healing rate in the infected wound was assessed using wound area, histopathological characteristics, and expression of growth factors including vascular endothelial growth factor (VEGF), transforming growth factor beta 1 (TGF-β1), and fibroblast growth factor 2 (FGF-2). Results: The wound area significantly decreased (p < 0.05) in the treated animals. There was a significant increase (p < 0.05) in new vessels, fibroblasts count, and collagen deposition in the ostrich oil-treated animals. Expression of VEGF, TGF-β1, and FGF-2 revealed the immunomodulation and angiogenesis effects of the ostrich oil on wound healing. Conclusions: Our study demonstrated that ostrich oil may be a useful treatment in infected cutaneous wounds.     

Fgf-18 is required for osteogenesis but not angiogenesis during long bone repair

Bone regeneration is a complex event that requires the interaction of numerous growth factors. Fibroblast growth factor (Fgf)-ligands have been previously described for their importance in osteogenesis during development. In the current study, we investigated the role of Fgf-18 during bone regeneration. By utilizing a unicortical tibial defect model, we revealed that mice haploinsufficient for Fgf-18 have a markedly reduced healing capacity as compared with wild-type mice. Reduced levels of Runx2 and Osteocalcin but not Vegfa accompanied the impaired bone regeneration. Interestingly, our data indicated that upon injury angiogenesis was not impaired in Fgf-18(+/-) mice. Moreover, other Fgf-ligands and Bmp-2 could not compensate for the loss of Fgf-18. Finally, application of FGF-18 protein was able to rescue the impaired healing in Fgf-18(+/-) mice. Thus, we identified Fgf-18 as an important mediator of bone regeneration, which is required during later stages of bone regeneration. This study provides hints on how to engineering efficiently programmed bony tissue for long bone repair.     

Antimicrobial peptides derived from growth factors

Growth factors, comprising diverse protein and peptide families, are involved in a multitude of developmental processes, including embryogenesis, angiogenesis, and wound healing. Here we show that peptides derived from HB-EGF, amphiregulin, hepatocyte growth factor, PDGF-A and PDGF-B, as well as various FGFs are antimicrobial, demonstrating a previously unknown activity of growth factor-derived peptides. The peptides killed the Gram-negative bacteria Escherichia coli, Pseudomonas aeruginosa, and the Gram-positive Bacillus subtilis, as well as the fungus Candida albicans. Several peptides were also active against the Gram-positive S. aureus. Electron microscopy analysis of peptide-treated bacteria, paired with analysis of peptide effects on liposomes, showed that the peptides exerted membrane-breaking effects similar to those seen after treatment with the “classical” human antimicrobial peptide LL-37. Furthermore, HB-EGF was antibacterial per se, and its epitope GKRKKKGKGLGKKRDPCLRKYK retained its activity in presence of physiological salt and plasma. No discernible hemolysis was noted for the growth factor-derived peptides. Besides providing novel templates for design of peptide-based antimicrobials, our findings demonstrate a previously undisclosed link between the family of growth factors and antimicrobial peptides, both of which are induced during tissue remodelling and repair.     

Low‐level laser therapy (780 nm) combined with collagen sponge scaffold promotes repair of rat cranial critical‐size defects and increases TGF‐β, FGF‐2, OPG/RANK and osteocalcin expression

The aim of this study was to evaluate the effect of collagen sponge scaffold (CSS) implantation associated with low‐level laser therapy (LLLT) on repairing bone defects. A single 5‐mm cranial defect was surgically created in forty Wistar rats, which then received one of the following four interventions (n = 10 per group): no treatment (G0); bone defect implanted with collagen sponge scaffold (CSS) alone (G1); defect treated with low‐level laser therapy (LLLT) (wavelength 780 nm; total energy density 120 J/cm²; power 50 mW) alone (G2); and CSS associated with LLLT treatment (G3). After surgery, animals in each group were euthanized at 21 days and 30 days (n = 5 per euthanasia time group). Bone formation was monitored by X‐ray imaging analysis. Biopsies were collected and processed for histological analysis and immunohistochemical evaluation of transforming growth factor‐beta (TGF‐β), fibroblast growth factor‐2 (FGF‐2), osteoprotegerin (OPG) and receptor activator of nuclear factor ? (RANK). Osteocalcin (OCN) was detected by immunofluorescence analysis. Compared to the G0 group, defects in the 30‐day G3 group exhibited increased bone formation, both by increase in radiopaque areas (P < 0.01) and by histomorphometric analysis (P < 0.001). The histopathological analysis showed a decreased number of inflammatory cells (P < 0.001). The combined CCS + LLLT (G3) treatment also resulted in the most intense immunostaining for OPG, RANK, FGF‐2 and TGF‐β, and the most intense and diffuse OCN immunofluorescent labelling at 30 days postsurgery (G3 vs. G0 group, P < 0.05). Therefore, the use of CCS associated with LLLT could offer a synergistic advantage in improving the healing of bone fractures.     

Bone Regeneration by Lactoferrin Released from a Gelatin Hydrogel

The objective of this study is to evaluate the potential of lactoferrin (LF), an iron-binding glycoprotein, to induce bone regeneration. A biodegradable gelatin hydrogel was prepared to allow LF release in vivo in a sustained fashion. When subcutaneously implanted into the back of mice, the gelatin hydrogel incorporating LF showed a longer LF retention period at the site of implantation than that of LF solution injection. An in vitro culture experiment with 3T3E1 cells (mouse-derived osteoblasts) revealed that the cells were proliferated to a significantly greater extent by the repeated addition of LF compared with a single addition of LF at the same dose. Following the implantation of gelatin hydrogels incorporating LF into a skull bone defect of rats, a significantly stronger bone regeneration at the defect was observed than in LF-free- or low-LF-treated rats. It is concluded that the sustained release from the gelatin hydrogels enables LF to enhance the in vivo activity of bone regeneration.