Fabrication of anodic and atomic layer deposition-alumina coated titanium implants for effective osteointegration applications.

Biomimicking the chemical, mechanical, and topographical properties of bone on an implant model is crucial to obtain rapid and effective osteointegration, especially for the large-area fractures of the skeletal system. Titanium-based biomaterials are more frequently preferred in clinical use in such cases and coating these materials with oxide layers having chemical/nanotopographic properties to enhance osteointegration and implantation success rates has been studied for a long time. The objective of this study is to examine the high and rapid mineralization potential of anodized aluminum oxide (AAO) coated and atomic layer deposition (ALD)-alumina coated titanium substrates on large deformation areas with difficult spontaneous healing. AAO-coated titanium (AAO@Ti) substrates were fabricated via anodization technique in different electrolytes and their osteogenic potential was analyzed by comparing them to the bare titanium surface as a control. In order to investigate the effect of the ionic characters gained by the surfaces through anodization, the oxidized nanotopographic substrates were additionally coated with an ultrathin alumina layer via ALD (ALD@AAO@Ti), which is a sensitive and conformal coating vapor deposition technique. Besides, a bare titanium sample was also coated with pure alumina by ALD (ALD@Ti) to investigate the effect of nanoscale surface morphology. XPS analysis after ALD coating showed that the ionic character of each surface fabricated by anodization was successfully suppressed. In vitro studies demonstrated that, among the substrates investigated, the mineralization capacity of MG-63 osteosarcoma cells were highest when incubated on ALD-treated and bare AAO@Ti samples that were anodized in phosphoric acid (H3PO4_AAO@Ti and ALD@H3PO4_AAO@Ti). Mineralization on these substrates also increased consistently beginning from day 2 to day 21. Moreover, immunocytochemistry for osteopontin (OPN) demonstrated the highest expression for ALD@H3PO4_AAO@Ti, followed by the H3PO4_AAO@Ti sample. Consequently, it was observed that, although ALD treatment improves cellular characteristics on all samples, effective mineralization requires more than a simple ALD coating or the presence of a nanostructured topography. Overall, ALD@H3PO4_AAO@Ti substrates can be considered as an implant alternative with its enhanced osteogenic differentiation potential and rapid mineralization capacity.

Bioinspired Collagen/Gelatin Nanopillared Films as a Potential Implant Coating Material.

Collagen-based Sharpey's fibers are naturally located between alveolar bone and tooth, and they have critical roles in a well-functioning tooth such as mechanical stability, facile differentiation, and disease protection. The success of Sharpey's fibers in these important roles is due to their unique location, vertical alignment with respect to tooth surface, as well as their micronanofiber architecture. Inspired by these structures, herein, we introduce the use of nanoporous anodic aluminum oxide molds in a drop-casting setup to fabricate biopolymeric films possessing arrays of uniform Collagen:Gelatin (Col:Gel) nanopillars. Obtained structures have diameters of ∼90 nm and heights of ∼300 nm, yielding significantly higher surface roughness values compared to their flat counterparts. More importantly, the nanostructures were parallel to each other but perpendicular to the underlying film surface imitating the natural collagenous structures of Sharpey's fibers regarding nanoscale morphology, geometrical orientation, as well as biochemical content. Viability testing showed that the nanopillared Col:Gel films have high cell viabilities (over 90%), and they display significantly improved attachment (ca. ∼ 2 times) and mineralization for Saos-2 cells when compared to flat Col:Gel films and Tissue Culture Polystyrene (TCPS) controls, plausibly due to their largely increased surface roughness and area. Hence, such Sharpey's fiber-inspired bioactive nanopillared Col:Gel films can be used as a dental implant coating material or tissue engineering platform with enhanced cellular and osteogenic properties.

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.

Patients- and tissue-specific bio-inks with photoactivated PRP and methacrylated gelatin for the fabrication of osteochondral constructs.

In osteochondral tissue engineering, while the biochemical and mechanical properties of hydrogels guide stem cell proliferation and differentiation, physical and chemical stimulators also affect the differentiation of stem cells. Herein, we presented a patient and tissue-specific strategy for the development of biomimetic osteochondral constructs with gradient compositions. Osteochondral constructs were fabricated by gradually printing of bio-inks consisting of therapeutic platelet-rich plasma (PRP), adipose tissue-derived mesenchymal stem cells (AdMSCs), and extracellular matrix (ECM) mimetic hydrogel, microwave-assisted methacrylated gelatin (Gel-MA). Periodic application of light in the near infrared region (600-1200 nm wavelength) was used to induce platelet activation and also AdMSCs' differentiation. Gel-MA has the same structure as type I collagen and PRP has cartilage tissue-specific bioactive components, so they provide the appropriate environment for the differentiation of AdMSCs to osteochondral tissue. Histology, immunocytochemistry, and biochemical analyses indicated enhanced glycosaminoglycan (GAG) and calcium content, mineralization, and ECM production. Furthermore, RT-PCR results indicated the expressions of bone- and cartilage-specific genes. In conclusion, the periodically photoactivated hydrogels with relatively low degradation rate and high mechanical strength, and tissue-specific biomimetic structure promoted in-vitro osteochondral tissue formation including hyaline and hypertrophic cartilage and bone phases.

Microwave assisted methacrylation of Kappa carrageenan: A bioink for cartilage tissue engineering.

In this study, we aimed to obtain stable Kappa carrageenan (κCar) hydrogel that could be used as a bioink for cartilage regeneration. For this purpose, we described an effective and considerably faster methacrylation process by using microwave energy. Thus, microwave-methacrylated κCar (Mw-κCar-MA) with ≥85% degree of methacrylation (DM) was synthesized despite the use of a low concentration of methacrylic anhydride (MA) at 1000 W in 5 min. Then, Mw-κCar-MA was photo-crosslinked by only using UV irradiation for 40 s. Characterization studies proved that Mw-κCar-MA hydrogels were stronger and have lower weight loss (~20% at 30 days) than that of conventionally synthesized κCar-MA hydrogels. Viscosities of the Mw-κCar-MA hydrogels were found to be sufficient to use in 3D bioprinters. Furthermore, Mw-κCar-MA hydrogels enhanced the viability, proliferation, and GAG deposition of ATDC5 chondrogenic cells. Therefore, we proposed that Mw-κCar-MA can be considered as a suitable bioink for cartilage tissue engineering.

Photo-activated platelet-rich plasma (PRP)-based patient-specific bio-ink for cartilage tissue engineering.

Nowadays, scientists focus on the development of tissue-specific and personalized bio-ink that can be used in 3D bioprinting technologies. Platelet-rich plasma (PRP) is a person-specific source that is used as a therapeutic adjunct for the treatment of cartilage damage because it offers a cocktail of growth factors that are necessary for wound healing and tissue regeneration. However, PRP treatments in the clinic are not satisfactory and require upgrading, especially the point of maintaining bioactivity. In this study, we presented PRP as a photo-activated and photo-crosslinkable bio-ink in terms of tissue-specific structures for the first time. We achieved long-term and constant rate growth factor release and bioactivity protection of PRP with satisfactory mechanical characteristics. Photo-crosslinked PRP hydrogel was enabled by the addition of microwave-induced methacrylated gelatin (Gel-MA), which is connected to platelets in PRP via integrin receptors in its structure and chemically cross-linked upon UV irradiation (300-500 nm). Photo-activation of PRP was realized by a polychromatic light source in the near-infrared region (PAC, 600-1200 nm). Our results showed that Gel-MA/PRP hydrogels with the desired mechanical properties (low degradation rate and high mechanical strength) released growth factors at a constant rate for the long-term by the periodic PAC application. In vitro cell culture studies (viability, proliferation, morphology, histology, immunochemistry, biochemistry, gene expression analyses) proved that proliferation and differentiation of the ATDC5 cells increased in the periodically light-applied Gel-MA/PRP hydrogel without any external chemical agents.

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.

Highly Methacrylated Gelatin Bioink for Bone Tissue Engineering.

Methacrylated gelatin (Gel-MA) is a commonly used biomaterial in bioprinting applications. The Gel-MA synthesis procedure is inadequate and needs to be improved, particularly from the point of optimization and efficacy. We report a significantly faster (by 5 min) and effective method to controllably synthesize Gel-MA using microwave energy (Mw at 1000 W power) with ≥90% degree of methacrylation (DM) even with the use of a very low concentration of methacrylic anhydride (MA). Rheological and mechanical analyses indicated that Gel-MA synthesized by Mw-assisted methacrylation enabled the formation of hydrogels that are more elastic and stronger and have a lower degradation rate (∼27% at 35 days) than Gel-MA synthesized by the conventional method. The viscosity values of the Gel-MA bioink were in the range applicable for use in 3D bioprinters. Additionally, Mw-assisted methacrylated Gel-MA hydrogels that have mechanically superior properties significantly enhanced the viability, attachment, proliferation, alkaline phosphatase (ALP) activity, mineral deposition, and mRNA expression levels of osteogenic genes of MC3T3-E1 preosteoblastic cells.

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.

Sustained release of 17ß-estradiol stimulates osteogenic differentiation of adipose tissue-derived mesenchymal stem cells on chitosan-hydroxyapatite scaffolds.

The aim of this study was to develop a 17ß-estradiol (E2)-releasing scaffold-nanoparticle system in order to promote osteogenic differentiation of rat adipose tissue-derived mesenchymal stem cells (AdMSCs) for bone tissue regeneration. E2-loaded poly(lactide-co-glycolide) (PLGA) nanoparticles with a diameter of ∼240 nm were produced via an emulsion-diffusion-evaporation method. Because of its higher encapsulation efficiency (54%), PLGA, which has a 65:35 composition, was chosen for the preparation of nanoparticles. Chitosan-hydroxyapatite (HA) scaffolds in macroporous structures with interconnected pores were prepared by combining microwave irradiation and gas-foaming techniques. PLGA nanoparticles were loaded onto scaffolds in 2 ways: via embedding after scaffold fabrication and during fabrication. While 100% of the loaded E2 was released during 55 days from scaffolds loaded by embedding, a controlled release behavior of E2 was observed over 135 days in scaffolds loaded during manufacture. The results of cell culture studies indicated that the controlled delivery of E2 from PLGA nanoparticles loaded on chitosan-HA scaffolds had a significant effect on the osteogenic differentiation of AdMSCs.

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.