Garlic extract enhances bioceramic bone scaffolds through upregulating ALP & BGLAP expression in hMSC-monocyte co-culture.

Bone homeostasis is predicated by osteoblast and osteoclast cell cycles where gene expressions are responsible for their differentiation from human mesenchymal stem cells (hMSC) and monocytes, respectively. The pro-osteogenic potential of an hMSC-monocyte co-culture can be measured through complementary DNA (mRNA synthesis) within the nucleus, known as quantitative polymerase chain reaction (qPCR). Through this technique, the effects of garlic extract (allicin) release from calcium phosphate bone scaffolds on gene expression of bone forming and bone remodeling cells was explored. Results show this complex biomaterial system enhances hMSC differentiation through the upregulation of bone-forming proteins. Osteoblastic gene markers alkaline phosphatase (ALP) and osteocalcin (BGLAP), are respectively upregulated by 3-fold and 1.6-fold by day 14. These mature osteoblasts then upregulate the receptor activator of nuclear factor-kB ligand (RANKL) which recruits osteoclast cells, as captured by a nearly 2-fold higher osteoclast expression of tartrate-resistance acid-phosphatase (ACP5). This also activates antagonist osteoprotegerin (OPG) expression in osteoblasts, decreasing osteoclast resorption potential and ACP5 expression by day 21. The pro-osteogenic environment with garlic extract release is further quantified by a 4× increase in phosphatase activity and visibly captured in immunofluorescent tagged confocal images. Also corroborated by enhanced collagen formation in a preliminary in vivo rat distal femur model, this work collectively reveals how garlic extract can enhance bioceramic scaffolds for bone tissue regenerative applications.

Ginger and Garlic Extracts Enhance Osteogenesis in 3D Printed Calcium Phosphate Bone Scaffolds with Bimodal Pore Distribution.

Natural medicines have long been used to treat physiological ailments where both ginger (gingerol) and garlic (allicin) are key players in immune system promotion, reduction in blood pressure, and lowering inflammation response. With their efficacy in bone healing, these compounds have great value as medicinal additives in bone scaffolds for localized treatment to support tissue formation, along with providing their natural therapeutic benefits. Utilization of 3D-printed (3DP) bone tissue engineering scaffolds as drug delivery vehicles for ginger and garlic extracts enables patient specificity in bone defect applications with enhanced osseointegration. Our objective is to understand their combined efficacy on osteogenesis when released from 3DP calcium phosphate bone scaffolds designed with a bimodal pore distribution. With a porous core and dense exterior, the resulting scaffolds have good mechanical integrity with 10 ± 1 MPa compressive strengths. Results show that ginger + garlic extracts released from bone scaffolds enhance their osteogenic potential through on site drug delivery. Both compounds exhibit exponential drug release profiles which fit Weibull distribution equations. The release of ginger extract also increases osteoblast proliferation by 59%. Both compounds show decreased osteoclast resorption activity, with a greater than 20% reduction in pit area on sample surfaces. Ginger + garlic extract induces a twofold increase in early osteoid tissue formation in vivo at week 4, in addition to a 30% increase in total bone area and a 90% increase in osteocytes with respect to control 3DP tricalcium phosphate scaffolds. Late-stage bone healing at week 10 reveals healthy angiogenic tissue, a twofold higher bone mineralization, and significant enhancement of type I collagen formation in the presence of ginger and garlic extracts. Naturally sourced ginger and garlic extracts provide osteogenic promotion and improved bone tissue in-growth in a patient-specific 3DP scaffold biomedical device for low load-bearing bone tissue engineering and dental applications.

Effects of Vitamin A (Retinol) Release from Calcium Phosphate Matrices and Porous 3D Printed Scaffolds on Bone Cell Proliferation and Maturation.

Vitamin A is a fat-soluble compound widely known for vision health. Highly variable reports on its effects on bone health have necessitated further research to truly understand its role on bone cell proliferation. Retinol, one bioactive form of vitamin A, is incorporated into synthetic bone graft scaffolds for low load-bearing clinical bone treatment. The objective of this work is to understand the effects of retinol on osteoblast and osteoclast cells when embedded within calcium phosphate matrices, including interconnected porous 3D printed tricalcium phosphate scaffolds. Results show that hydrophobic retinol can be released from bone scaffolds when a combination of biodegradable polymers, polycaprolactone and polyethylene glycol, are employed as drug carriers. The release of retinol in vitro can support a 20 ± 1% increase in osteoblast (bone-forming) cell proliferation with proper cell adhesion and filopodial extensions. Osteoclast cell morphology is necrosed and torn with a reduction in proliferation at approximately 6 ± 1% when retinol is present. In addition, inhibition of osteoclastic resorption pit bays is noted using scanning electron microscopy. With the scaffolds' round pore interconnectivity facilitating retinol release, this system can provide an alternative to traditional bone grafts while additionally supporting bone healing through enhanced osteoblast cell proliferation and inhibition of osteoclast resorption activity.

Effects of surface area and topography on 3D printed tricalcium phosphate scaffolds for bone grafting applications.

Additive manufacturing (AM), or 3D printing, of bioceramic scaffolds promises personalized treatment options for patients with site-specific designability for repair and reconstruction of bone defects. Although the theory for creating these complex geometries has already been made possible through AM's advancement, such shapes' manufacturability is difficult due to printing with ceramics' inherent complexities. Ceramics have the added challenge of being highly brittle, poor handleability of green (pre-sintered) parts, making complex shape high strength parts challenging to manufacture. This has led to a significant literature gap regarding the feasibility of creating bioceramic scaffolds with unique architectures that can be used in site-specific, individualized patient treatment. This work aims to successfully create complex topographical surfaces of cylindrical bone-like scaffolds to understand the correlation of increasing the scaffold surface area on mechanical properties and in vitro osteoblast cell proliferation. An increase in osteoblast cell proliferation and facilitation in cellular attachment can ultimately lead to improved bone healing. This work explores the printing parameters within an Innovent+® ExOne binder jet 3D printer to produce scaffold designs from synthesized tricalcium phosphate powder. Mechanical testing reveals the designed structures enhance scaffold compressive strength by 30% compared to control dense cylindrical scaffolds. Osteoblast cell proliferation is also increased due to changes in surface topography with a nearly 2-fold increase. Our work incorporates macro-level topographical changes to increase surface area, which is another avenue that could be combined with other scaffold features such as porosity. Results show bulk surface topography modifications via 3D printing can increase surface area to support enhanced biological response without compromising mechanical properties. This discovery may enable a future generation of porous scaffolds with external structures for further progress towards proper defect-specific synthetic bone grafts.

Natural Antibiotic Oregano in Hydroxyapatite-Coated Titanium Reduces Osteoclastic Bone Resorption for Orthopedic and Dental Applications.

Traditional infection prevention and treatment methods include synthetic antibiotics, which can cause severe adverse side effects. Carvacrol and thymol are biologically active monoterpenoid extractants from oregano leaves with antibiotic capabilities; however, little is known regarding their effects on bone tissue engineering. The objective of this work is to understand their effects on osteogenesis, specifically with osteoblast and osteoclast cells, from surface-modified Ti6Al4V with plasma sprayed hydroxyapatite (HA) coatings. This system is an alternative to cemented implants to aid in bone healing. Results reveal that full carvacrol release from the HA matrix is successful in aqueous environments and modulation of release kinetics can also be made using polycaprolactone (PCL) and polyethylene glycol (PEG) polymers. From HA-pressed disc samples in physiological pH, full carvacrol release is achieved in 10 days using PCL/PEG, about 95% release in 50 days using no polymer, and 60% in 50 days when using a PCL coating. Without polymer, full carvacrol release is achieved after 3 days from HA coatings in both physiological pH and acidic pH, mimicking the post-surgery environment. The release is assessed as a diffusion-based mechanism in phosphate-buffered saline but degradation-based mechanism in acetate buffer solution. Carvacrol and thymol show bacterial inhibition of Staphylococcus epidermidis and no cytotoxic effects on osteoblast proliferation in vitro. Carvacrol and thymol also induce a significant 7% reduction in osteoclast tartrate-resistant acid phosphatase (TRAP) activity, caused by poorly attached cellular morphologies, leading to an approximately 65% reduction in osteoclast resorption pit formation. Our goal is to demonstrate a natural medicinal system that can support bone healing while providing infection prevention and reducing costly revision surgeries for orthopedic and dental applications.

Effects of chitosan-loaded hydroxyapatite on osteoblasts and osteosarcoma for chemopreventative applications.

Osteosarcoma remains one of the most common malignant primary bone tumors. Post-surgical defect repair combined with tumor suppression remains a major clinical challenge. Investigations of alternative treatments for osteosarcoma, while promising, have led to multi-drug resistance. These constraints of common treatment strategies have triggered the need for new therapeutic candidates in bone cancer treatment. Chitosan, a common biopolymer utilized in bone and tissue engineering applications, has recently been studied as a pro-apoptotic agent in metastatic cell lines like breast cancer, but has not been utilized in bone cancer applications. In this study, chitosan was directly loaded onto HA disks to evaluate its in vitro release and effects on human fetal osteoblast (hFOB) and human osteosarcoma (MG-63) cell lines. It is hypothesized that the sustained release of chitosan will decrease osteosarcoma cell proliferation and enhance proliferation of osteoblast cells. Through morphological characterization and MTT assay analysis, chitosan showed no toxicity to human fetal osteoblast (hFOB) cells. Chitosan was also shown to decrease human osteosarcoma cell viability by up to 96% compared to control samples. This suggests a pro-apoptotic mechanism against osteosarcoma as well as the potential clinical application of chitosan as a drug candidate in ceramic scaffolds at tumor resected sites.

Thermal Oxide Layer Enhances Crystallinity and Mechanical Properties for Plasma-Sprayed Hydroxyapatite Biomedical Coatings.

The stability of plasma-sprayed hydroxyapatite (HA) coatings on metallic implants in vivo remains a significant challenge for load-bearing orthopedic implants despite their excellent mechanical and osteoconductive properties. This study focuses on oxide layer formation on the surface of Ti6Al4V samples through furnace heating at 600, 700, and 800 °C for 10 min for optimization of the most effective oxide layer to increase plasma coating crystallinity and improve plasma coating bond strength with the metal surface. The 800 °C heat treatment shows an effective oxide layer which increases coating crystallinity from 64 to 75% and coating adhesive bond strength from 25.9 ± 2.3 to 30.7 ± 1.1 MPa, while simultaneously reducing the dissolution rate of HA coatings. The addition of biologically relevant dopants, MgO and SiO2, show negligible effects on crystallinity and adhesive bond strength on plasma-sprayed HA coatings and additionally show an enhancement effect on osteoblast proliferation and differentiation. Moreover, the inclusion of these additivess shows an increase in osteogenesis in a rat distal femur model after 6 and 10 weeks of implantation. Overall, this study provides a direct solution to improve the crystallinity, adhesive bond strength, and osteogenic properties of plasma-sprayed HA coatings on orthopedic implants that is more manufacturable and translational from research to an industrial scale.

Vitamin D3 Release from Traditionally and Additively Manufactured Tricalcium Phosphate Bone Tissue Engineering Scaffolds.

Bone is a randomized, complex porous network which researchers have tried to mimic within bone tissue engineering scaffolds. The objective of this study was to understand the effects of random and controlled scaffold porosity on the release kinetics of vitamin D3 to determine if a designed porous structure was comparable in effectiveness on osteoblast proliferation to the randomized essence of natural bone. In this study, porous tricalcium phosphate (TCP) scaffolds were prepared by fugitive material removal method using naphthalene and 3D printing to model random and controlled porosity, respectively. Scaffold comparison was made based on open pore volume percentage of which naphthalene scaffolds had 45.8 ± 1.5% and 3D printed scaffolds had 48.9 ± 2.5%, Comparative analysis of traditional bioceramic processing to additive manufacturing is limited especially regarding drug release kinetics. Results showed the naphthalene scaffold surface area was only 0.3% that of 3D printed scaffolds due to the lower open pore interconnectivity. This increase in surface area produced higher release of drug and osteoblast proliferation in 3D printed scaffolds comparatively. By 11 days, osteoblast proliferation was enhanced by 64% from scaffolds manufactured using 3D printing compared to traditional processing. Understanding the effects of processing methods of TCP scaffolds on the release kinetics of vitamin D3 and the system effects on cells can aid in low load bearing applications for bone tissue engineering.

Clinical significance of three-dimensional printed biomaterials and biomedical devices.

Three-dimensional printing (3DP) is becoming a standard manufacturing practice for a variety of biomaterials and biomedical devices. This layer-by-layer methodology provides the ability to fabricate parts from computer-aided design files without the need for part-specific tooling. Three-dimensional printed medical components have transformed the field of medicine through on-demand patient care with specialized treatment such as local, strategically timed drug delivery, and replacement of once-functioning body parts. Not only can 3DP technology provide individualized components, it also allows for advanced medical care, including surgical planning models to aid in training and provide temporary guides during surgical procedures for reinforced clinical success. Despite the advancement in 3DP technology, many challenges remain for forward progress, including sterilization concerns, reliability, and reproducibility. This article offers an overview of biomaterials and biomedical devices derived from metals, ceramics, polymers, and composites that can be three-dimensionally printed, as well as other techniques related to 3DP in medicine, including surgical planning, bioprinting, and drug delivery.

Mechanical and biological properties of ZnO, SiO2, and Ag2O doped plasma sprayed hydroxyapatite coating for orthopaedic and dental applications.

In this study, we explored a ternary dopant system utilizing 0.25 wt% ZnO to induce osteogenesis, 0.5 wt% SiO2 to induce angiogenesis, and 2.0 wt% Ag2O to provide secondary infection control within a plasma assisted hydroxyapatite coating for orthopaedic or dental applications. The objective of this study was to understand the effects of ZnO, SiO2, and Ag2O dopants on the mechanical and biological properties of hydroxyapatite (HA) coatings on titanium (Ti). Coatings were deposited using a 30 kW plasma spray system equipped with a supersonic nozzle to produce above standard coating bond strengths of 24 ±â€¯2 MPa on Ti6Al4V and 22 ±â€¯1 MPa on commercially pure Ti substrates. Antibacterial properties were revealed in vitro against E. coli and S. aureus. The ternary dopant system was implanted in 18 male Sprague-Dawley rats with timepoints of 5 and 10 weeks. By week 5, ZnSiAg-HA produced 32% bone mineralization of 68% total bone formation compared to only 11% bone mineralization of 55% total bone formation in the undoped coating. This system can be employed for replacement surgeries and revision surgeries to reduce healing time and enhance osseointegration. STATEMENT OF SIGNIFICANCE: Total hip replacements increased 124% from 2000 to 2010 with an ever-increasing rate due to the rise in average life span and an escalation in surgeries for young patients. Replacement surgeries come with the risk of rejection, poor integration, and infection. This study incorporates biologically relevant metallic oxides of ZnO, SiO2, and Ag2O within a hydroxyapatite coating on titanium deposited using a radio frequency induction plasma spray. A ternary dopant system has not been explored in the current literature and little is known about these particular dopants in vivo. This proposed system can be employed for replacement surgeries to lower healing time and enhance osseointegration between implant and host tissue.

Effects of vitamin D3 release from 3D printed calcium phosphate scaffolds on osteoblast and osteoclast cell proliferation for bone tissue engineering.

Vitamin D3 is a hydrophobic micronutrient and is known for inhibiting osteoclastic bone resorption in vivo via suppression of the Receptor Activator of Nuclear factor-Kappa B (RANK ligand) expression in osteoblasts. Although vitamin D is well-known for its promotion in bone health, little is known on its effects directly on bone cells. The objective of this study was to understand the effects of vitamin D3 release from 3D printed calcium phosphate scaffolds towards bone cell proliferation. In this study, cholecalciferol, a common intake form of vitamin D3, was successfully able to release from the scaffold matrix via the use of polyethylene glycol. Results showed a decrease in osteoclast resorption pits and healthier osteoblast cellular morphology compared to the control. Additively manufactured tricalcium phosphate scaffolds with designed porosity were loaded with vitamin D3 and showed controlled release profiles in phosphate buffer and acetate buffer solutions. The release kinetics of vitamin D3 from calcium phosphate scaffolds enabling osteoblast proliferation and inhibiting osteoclastic resorption can enhance healing for low load bearing applications for bone defects or permeate voids left by tumor resection.

Compositionally graded doped hydroxyapatite coating on titanium using laser and plasma spray deposition for bone implants.

Plasma sprayed hydroxyapatite (HA) coating is known to improve the osteoconductivity of metallic implants. However, the adhesive bond strength of the coating is affected due to a mismatch in coefficients of thermal expansion (CTE) between the metal and HA ceramic. In this study, a gradient HA coating was prepared on Ti6Al4V by laser engineered net shaping (LENS™) followed by plasma spray deposition. In addition, 1 wt% MgO and 2 wt% Ag2O were mixed with HA to improve the biological and antibacterial properties of the coated implant. Results showed that the presence of an interfacial layer by LENS™ enhanced adhesive bond strength from 26 ±â€¯2 MPa for just plasma spray coating to 39 ±â€¯4 MPa for LENS™ and plasma spray coatings. Presence of MgO and Ag2O did not influence the adhesive bond strength. Also, Ag+ ions release dropped by 70% less with a gradient HA LENS™ layer due to enhanced crystallization of the HA layer. In vitro human osteoblast cell culture revealed presence of Ag2O had no deleterious effect on proliferation and differentiation when compared to pure HA as control and provided antibacterial properties against E. coli and S. aureus bacterial strands. This study presents an innovative way to improve interfacial mechanical and antibacterial properties of plasma sprayed HA coating for load-bearing orthopedic as well as dental implants. STATEMENT OF SIGNIFICANCE: Implants are commonly composed of metals that lack osteoconductivity. Osteoconductivity is a property where bone grows on the surface meaning the material is compatible with the surrounding bone tissue. Plasma sprayed hydroxyapatite (HA) coating improves the osteoconductivity of metallic implants, however, the adhesive bond strength can be weak. This study incorporates a gradient HA coating by using an additive manufacturing technique, laser engineered net shaping (LENS™), followed by plasma spray deposition to enhance the adhesive bond strength by incorporating a thermal barrier. The proposed system has not been well studied in the current literature and the results presented bring forth an innovative way to improve the interfacial mechanical and antibacterial properties of plasma sprayed HA coating for load-bearing orthopedic implants.

Effects of polycaprolactone on alendronate drug release from Mg-doped hydroxyapatite coating on titanium.

The scientific objective of this study was to understand the influence of PCL coating on alendronate drug release kinetics in vitro. Our hypothesis was PCL coating would minimize burst release of alendronate from plasma sprayed Mg-doped hydroxyapatite (HA) coated commercially pure titanium (CpTi) samples. In the US alone, over 44 million women and men aged 50 and older are affected by osteoporosis which can lead to replacement and/or revision surgeries. Alendronate is a widely-used drug for treating osteoporosis and would be an ideal drug to be loaded and released from these replacement systems. Initial burst release is a common phenomenon for the most drug loaded devices. To modulate the release kinetics, a biodegradable polymer, polycaprolactone (PCL), coating with slow degradable kinetics was employed. Samples with 2 and 4 wt% PCL showed about 34% and 26% release of alendronate within the first 24 h, respectively, compared to 75% burst release without any PCL coating. With the addition of a PCL coating, a controlled release kinetics of alendronate was achieved from HA coated titanium implants, which can potentially impact millions of patients worldwide having compromised bone due to osteoporosis.