UiO-66 metal-organic framework as a double actor in chitosan scaffolds: Antibiotic carrier and osteogenesis promoter.

Metal-organic frameworks (MOFs) have recently emerged as a useful class of nanostructures with well-suited characteristics for drug delivery applications, due to the high surface area and pore size for efficient loading. Despite their use as a nano-carrier for controlled delivery of various types of drugs, the inherent osteo-conductive properties have stolen a great attention as a growing area of investigation. Here, we evaluated the double function of UiO-66 MOF structure as a carrier for fosfomycin antibiotic and also as an osteogenic differentiation promoter when introduced in 3D chitosan scaffolds, for the first time. Our results revealed that the wet-spun chitosan scaffolds containing fosfomycin loaded UiO-66 nanocrystals (CHI/UiO-66/FOS) possessed fiber mesh structure with integrated micro-scale fibers and increased mechanical strength. In vitro antibacterial studies indicated that CHI/UiO-66/FOS scaffolds showed bactericidal activity against Staphylococcus aureus. Moreover, the scaffolds were biocompatible to MC3T3-E1 pre-osteoblasts and significantly up-regulated the expression of osteogenesis-related genes and facilitated the extracellular matrix mineralization, in vitro. Taken together, our results demonstrate UiO-66 MOFs can present double functionality and CHI/UiO-66/FOS scaffolds hold a significant potential to be further explored as an alternative approach in treating infected bone defects like osteomyelitis.

Photobiomodulation combined with adipose-derived stem cells encapsulated in methacrylated gelatin hydrogels enhances in vivo bone regeneration.

Reconstruction of bone defects is still a significant challenge. The aim of this study was to evaluate the effect of application of photobiomodulation (PBM) to enhance in vivo bone regeneration and osteogenic differentiation potential of adipose-derived stem cells (ADSCs) encapsulated in methacrylated gelatin (GEL-MA) hydrogels. Thirty-six Sprague-Dawley rats were randomly separated into 3 experimental groups (n = 12 each). The groups were control/blank defect (I), GEL-MA hydrogel (II), and ADSC-loaded GEL-MA (GEL-MA+ADSC) hydrogel (III). Biparietal critical sized bone defects (6 mm in size) are created in each animal. Half of the animals from each group (n = 6 each) were randomly selected for PBM application using polychromatic light in the near infrared region, 600-1200 nm. PBM was administered from 10 cm distance cranially in 48 h interval. The calvaria were harvested at the 20th week, and macroscopic, microtomographic, and histologic evaluation were performed for further analysis. Microtomographic evaluation demonstrated the highest result for mineralized matrix formation (MMF) in group III. PBM receiving samples of group III showed mean MMF of 79.93±3.41%, whereas the non-PBM receiving samples revealed mean MMF of 60.62±6.34 % (p=0.002). In terms of histologic evaluation of bone defect repair, the higher scores were obtained in the groups II and III when compared to the control group (2.0 for both PBM receiving and non-receiving specimens; p<0.001). ADSC-loaded microwave-induced GEL-MA hydrogels and periodic application of photobiomodulation with polychromatic light appear to have beneficial effect on bone regeneration and can stimulate ADSCs for osteogenic differentiation.

Sustained release of growth factors from photoactivated platelet rich plasma (PRP).

The goal of this study is to specify the ability of polychromatic light source (PAC), providing effective wavelengths in the range of 600-1200 nm (near-infrared region, NIR), to activate human platelets in platelet-rich plasma (PRP) and to achieve sustained and controlled release of growth factors from photoactivated platelets. PRP was isolated from human blood and treated with PAC in different time intervals during 1, 5 and 10 min from 10 cm distance to the platelets. ATP secretion and then, calcium release from platelets significantly increased after light application. Photostimulation of platelets triggered lamellipodia extension, numerous filopodia formation, and platelet agglomeration as activation indicators. P-selectin expression was significantly increased after the application of PAC. In conclusion, PRP was successfully activated with PAC for 10 min and realized activation-dependent sustained growth factor release during 28 days. We proved that PAC which has a great potential of activation of PRP enables sustained growth factor release from PRP with a periodic use for therapeutic applications of PRP.

Photobiomodulation with polychromatic light increases zone 4 survival of transverse rectus abdominis musculocutaneous flap.

OBJECTIVE: The aim of this study was to evaluate the effect of relatively novel approach of application of polychromatic light waves on flap survival of experimental musculocutaneous flap model and to investigate efficacy of this modality as a delay procedure to increase vascularization of zone 4 of transverse rectus abdominis musculocutaneous (TRAM) flap. METHODS: Twenty-one Wistar rats were randomized and divided into 3 experimental groups (n = 7 each). In group 1 (control group), after being raised, the TRAM flap was sutured back to its bed without any further intervention. In group 2 (delay group), photobiomodulation (PBM) was applied for 7 days as a delay procedure, before elevation of the flap. In group 3 (PBM group), the TRAM flap was elevated, and PBM was administered immediately after the flap was sutured back to its bed for therapeutic purpose. PBM was applied in 48 hours interval from 10 cm. distance to the whole abdominal wall both in groups 2 and 3 for one week. After 7 days of postoperative follow-up, as the demarcation of necrosis of the skin paddle was obvious, skin flap survival was further evaluated by macroscopic, histological and microangiographic analysis. RESULTS: The mean percentage of skin flap necrosis was 56.17 ± 23.68 for group 1, 30.92 ± 17.46 for group 2 and 22.73 ± 12.98 for group 3 PBM receiving groups 2 and 3 revealed less necrosis when compared to control group and this difference was statistically significant. Vascularization in zone 4 of PBM applied groups 2 and 3 was higher compared to group 1 (P = 0.001). Acute inflammation in zone 4 of group 1 was significantly higher compared to groups 2 and 3 (P = 0.025). Similarly, evaluation of zone 1 of the flaps reveled more inflammation and less vascularization among the samples of the control group (P = 0.006 and P = 0.007, respectively). Comparison of PBM receiving two groups did not demonstrate further difference in means of vascularization and inflammation density (P = 0.259). CONCLUSION: Application of PBM in polychromatic fashion enhances skin flap survival in experimental TRAM flap model both on preoperative basis as a delay procedure or as a therapeutic approach. Lasers Surg. 51:538-549, 2019. © 2019 Wiley Periodicals, Inc.

A bioprintable form of chitosan hydrogel for bone tissue engineering.

Bioprinting can be defined as 3D patterning of living cells and other biologics by filling and assembling them using a computer-aided layer-by-layer deposition approach to fabricate living tissue and organ analogs for tissue engineering. The presence of cells within the ink to use a 'bio-ink' presents the potential to print 3D structures that can be implanted or printed into damaged/diseased bone tissue to promote highly controlled cell-based regeneration and remineralization of bone. In this study, it was shown for the first time that chitosan solution and its composite with nanostructured bone-like hydroxyapatite (HA) can be mixed with cells and printed successfully. MC3T3-E1 pre-osteoblast cell laden chitosan and chitosan-HA hydrogels, which were printed with the use of an extruder-based bioprinter, were characterized by comparing these hydrogels to alginate and alginate-HA hydrogels. Rheological analysis showed that all groups had viscoelastic properties. It was also shown that under simulated physiological conditions, chitosan and chitosan-HA hydrogels were stable. Also, the viscosity values of the bio-solutions were in an applicable range to be used in 3D bio-printers. Cell viability and proliferation analyses documented that after printing with bio-solutions, cells continued to be viable in all groups. It was observed that cells printed within chitosan-HA composite hydrogel had peak expression levels for early and late stages osteogenic markers. It was concluded that cells within chitosan and chitosan-HA hydrogels had mineralized and differentiated osteogenically after 21 days of culture. It was also discovered that chitosan is superior to alginate, which is the most widely used solution preferred in bioprinting systems, in terms of cell proliferation and differentiation. Thus, applicability and printability of chitosan as a bio-printing solution were clearly demonstrated. Furthermore, it was proven that the presence of bone-like nanostructured HA in alginate and chitosan hydrogels improved cell viability, proliferation and osteogenic differentiation.

A Biomimetic Alternative to Synthetic Hydroxyapatite: "Boron-Containing Bone-Like Hydroxyapatite" Precipitated From Simulated Body Fluid.

BACKGROUND: Biological hydroxyapatite (HA), has several mechanical and physical advantages over the commercially available synthetic apatite (CAP-HA). The aim of this in vivo study was to investigate the effect of osteoinductive "bone-like hydroxyapatite" obtained from simulated body fluid (SBF) combined with osteoinductive "boron" (B) on bone healing. MATERIALS: Bone like nanohydroxyapatite (SBF-HA) was precipitated from 10× simulated body fluid (10×SBF). Thirty Sprague-Dawley rats were randomly divided into 5 experimental groups (n = 6 each). The groups were involving blank defect, chitosan, SBF-HA, SBF-HA/B, and CAP-HA. Two biparietal round critical sized bone defect was created using a dental burr. The rats were sacrificed respectively at the end of second and fourth months after surgery and their calvarium were harvested for further macroscopic, microtomographic, and histologic evaluation. RESULTS: The SBF-HA/B group demonstrated the highest mineralized matrix formation rates (30.69 ± 3.73 for the second month, 62.68 ± 7.03 for the fourth month) and was significantly higher than SBF-HA and the CAP-HA groups. The SBF-HA/B group demonstrated the highest mineralized matrix formation rates (30.69 ± 3.73 for the second month, 62.68 ± 7.03 for the fourth month) and was significantly higher than SBF-HA and the CAP-HA groups. In means of bone defect repair histologically, the highest result was observed in the SBF-HA/B group (P < 0.001). CONCLUSIONS: The "bone-like hydroxapatite" obtained from simulated body fluid is worth attention when both its beneficial effects on bone healing and its biological behavior is taken in consideration for further bone tissue engineering studies. It appears to be a potential alternative to the commercially available hydroxyapatite samples.

A Bio-Acoustic Levitational (BAL) Assembly Method for Engineering of Multilayered, 3D Brain-Like Constructs, Using Human Embryonic Stem Cell Derived Neuro-Progenitors.

A bio-acoustic levitational assembly method for engineering of multilayered, 3D brainlike constructs is presented. Acoustic radiation forces are used to levitate neuroprogenitors derived from human embryonic stem cells in 3D multilayered fibrin tissue constructs. The neuro-progenitor cells are subsequently differentiated in neural cells, resulting in a 3D neuronal construct with inter and intralayer neurite elongations.

Microwave-assisted fabrication of chitosan-hydroxyapatite superporous hydrogel composites as bone scaffolds.

In this study, a novel scaffold fabrication method was developed by combining microwave irradiation and gas foaming. Chitosan superporous hydrogels (SPHs) and chitosan-hydroxyapatite (HA) superporous hydrogel composites (SPHCs) were prepared by using this method in the presence of crosslinking agent, glyoxal, and a gas-blowing agent, NaHCO3. In order to examine the effect of HA on composite structure and cellular behaviour, two types of HA particles, i.e. spherical beads in 45-80 µm diameter and powder form, were used. While rapid heating with microwave irradiation enhances gas blowing, pH increment, which is accelerated by NaHCO3 decomposition, provides better crosslinking. Thus, interconnected and well-established macroporous hydrogels/hydrogel composites were produced easily and rapidly (~1 min). Cell culture studies, which were carried out under static and dynamic conditions with MC3T3-E1 pre-osteoblastic cells, indicated that chitosan-HA bead SPHCs supported cellular proliferation and osteoblastic differentiation better than chitosan SPHs and chitosan-HA powder SPHCs. In conclusion, simultaneous gas foaming and microwave crosslinking can be evaluated for the preparation of composite scaffolds which have superior properties for bone tissue engineering.

Estrogen as a novel agent for induction of adipose-derived mesenchymal stem cells for osteogenic differentiation: in vivo bone tissue-engineering study.

BACKGROUND: This study investigated whether the in vivo osteogenic differentiation potential of adipose-derived mesenchymal stem cells is enhanced by 17ß-estradiol. METHODS: Thirty Sprague-Dawley rats were randomized and divided into five experimental groups. For the surgical procedure, biparietal full-thickness bone defects (7 mm in diameter) were created. A chitosan-hydroxyapatite scaffold was used as the vehicle system for 17ß-estradiol-loaded nanoparticles and adipose-derived mesenchymal stem cells. The first group, the blank defect group, was the control group. The defects were filled with either scaffold, estradiol, and scaffold; scaffold and adipose-derived mesenchymal stem cells; or estradiol, scaffold, and adipose-derived mesenchymal stem cells as experimental groups. The rats were killed at the end of weeks 4 and 12, and their calvariae were harvested for histologic and microtomographic evaluation. RESULTS: Micro-computed tomographic evaluation of estradiol, scaffold, and adipose-derived mesenchymal stem cells revealed the highest median value (82.59 ± 17.17), and the difference was significant compared with the blank defect group (p = 0.004). Histologic samples demonstrated a significant difference between experimental groups for bone defect repair at the end of weeks 4 and 12 (p = 0.003 and p < 0.001). The estradiol, scaffold, and adipose-derived mesenchymal stem cell group had the highest median score (3.00 ± 0.0) at week 12, which was significantly higher than scores for the scaffold and adipose-derived mesenchymal stem cell group and the blank defect group. CONCLUSION: 17ß-Estradiol appears to be a novel and promising agent for future cell-based bone tissue-engineering studies.

Evaluation of L929 fibroblast attachment and proliferation on Arg-Gly-Asp-Ser (RGDS)-immobilized chitosan in serum-containing/serum-free cultures.

In this study, chitosan membranes prepared by the solvent casting method were modified with the Arg-Gly-Asp-Ser (RGDS) sequence of fibronectin using the photochemical immobilization technique. The results obtained from attenuated total reflection-Fourier transform infrared spectra and X-ray photoelectron spectroscopy studies confirmed the successful immobilization of RGDS on chitosan membranes. The immobilized peptide concentration was determined by ninhydrin analysis on the order of 10(-7) mol/cm(2). In vitro cell culture studies were performed with L929 mouse fibroblasts to investigate the effect of biomodification on fibroblast cell behaviour in serum-free and 10% serum-containing media. The results obtained from cell culture studies pointed out the specific interactions between biosignal RGDS molecules and fibroblast cells. A triggered cell attachment and proliferation were observed on RGDS-modified chitosan membranes that were more distinguishable in serum-free medium. In addition, the photochemical immobilization technique was realized in the presence of a photomask that was used to immobilize the RGDS molecules in a defined micropattern. L929 mouse fibroblasts attached on the RGDS-micropatterned areas indicating integrin-mediated interactions.