Rapid hepatic perfusion decellularization: technique and critique.

BACKGROUND: Organ shortage facing the increasing success of liver transplantation has provoked research into the utilization of animal organs for clinical transplantation. The technique of whole-organ decellularization aims at the removal of the antigenic cellular content, thus evading the immune rejection cascade and the production of complex three-dimensional extracellular matrices of the entire organs with preservation of their intrinsic vascular networks rendering them transplantable. The aim of this study was the production of decellularized rabbit liver matrices by applying a simple, rapid perfusion decellularization technique and their characterization (both qualitatively and quantitatively). MATERIALS AND METHODS: Decellularization of the caudate hepatic lobes of New Zealand white rabbits (n = 22) was achieved through sequential perfusion of the portal venous system with deionized water, 0.8% Triton X-100 and 0.8% sodium dodecyl sulphate (SDS). Decellularized specimens were characterized both qualitatively (histology, fluoroscopy, corrosion casting and scanning electron microscopy) and quantitatively (total collagen assay [colorimetric] and total DNA assay [Hoechst 33258]). A Student's t-test was used to compare quantitative laboratory results before and after decellularization. A probability (P) value of <0.05 was considered significant. RESULTS: Effective decellularization was achieved as proven by histology and quantitative assessment (DNA remnants <1.5%, P = 0.0009), while preserving 68% of the total collagen content (P = 0.003). Portal vascular network integrity was confirmed by fluoroscopy and corrosion casting. Scanning electron microscopy also confirmed the preservation of the three-dimensional architecture. CONCLUSIONS: Liver perfusion decellularization technique using both 0.8% Triton X-100 and 0.8% SDS is a simple and rapid technique, yielding efficiently decellularized liver matrices preserving their vascular integrity, 3D architecture and 68% of total collagen content.

Enhancing mandibular bone regeneration and perfusion via axial vascularization of scaffolds.

OBJECTIVE: Reconstruction of large and complex bone segments is a challenging problem facing maxillofacial surgery. The majority of current regenerative approaches rely on extrinsic vascularization, which is deficient after cancer ablation and irradiation. The aim of the study was to investigate the efficacy of intrinsic axial vascularization of synthetic bone scaffolds in the management of critical-size mandibular defects. MATERIALS AND METHODS: Scaffold-guided mandibular regeneration in two groups of adult male goats was compared. Only the scaffolds of the second group were axially vascularized via in situ embedding of an arteriovenous loop through microsurgical anastomosis of facial vessels. After 6 months of follow up, both groups were compared through radiological, biomechanical, histological and histomorphometric analysis. RESULTS: The axially vascularized constructs have showed significantly more central vascularization (p = 0.021) and markedly enhanced central bone formation (p = 0.08). The biomechanical characteristics were enhanced, but the difference between both groups was not statistically significant (p = 0.98). CONCLUSIONS: Axially vascularized synthetic mandibular grafts show better vascularization at their central regions, permitting more efficient bone regeneration. CLINICAL RELEVANCE: The encouraging results of the proposed technique could be of benefit in optimizing the reconstruction of large critical-size bone defects.

Navigating the future of guided dental implantology: A scoping review.

BACKGROUND: The aim of this scoping review was to understand the development of robotics and its accuracy in placing dental implants when compared to other forms of guided surgery. METHODS: An electronic search was conducted on the electronic databases of PubMed, Cochrane, and Science direct with the following queries: ((robotics) AND (dental implant)) AND (accuracy). The search timeline was between 2017 and 2022. RESULTS: A total of 54 articles were screened for title and abstract, of which 16 were deemed eligible for inclusion. Thirty-one articles were excluded mainly because they were out of topic (not relevant) or not in English. In total, 16 articles were included for analysis. CONCLUSIONS: This review thoroughly analyses 5 years of literature concerning the evolution of robotics in dental implant surgery, underscoring the necessity for additional research on nascent technologies reported and a comparative study with static and dynamic systems for clinical efficacy evaluation.

Micro/Nanostructured Bioactive Titanium Implant Surface with Sol-Gel Silicate Glass Nanoparticles.

PURPOSE: To develop a surface coating of sol-gel 70S30C bioactive glass (BAG) nanoparticles on titanium disks and dental implants and characterize the BAG coating from the standpoint of average surface roughness, adhesion strength, and coating stability upon implant insertion under clinical settings. MATERIALS AND METHODS: BAG was prepared using a modified sol-gel technique, then milled into nanoparticles. The resultant powder was characterized in terms of phase structure, composition, and particle size. Titanium disks and dental implants were coated with BAG nanoparticles via electrophoretic deposition. Surface characterization of coated implants was conducted. Uncoated and BAGcoated implants were examined for average surface roughness using a confocal laser scanning microscope. Pull-off tests were conducted to measure the adhesion strength of the BAG coating to the underlying disks. To measure the amount of coating loss and evaluate the effect of insertion on coating thickness, coated implants were inserted under clinical settings into artificial and natural bones. RESULTS: BAG nanoparticles had an amorphous structure with particle sizes < 20 nm in diameter. Electrophoresis resulted in a continuous coating that covered the whole implant surface. Microscopic analysis confirmed the porous nanostructure of the BAG coating, which formed a homogenous surface with microcracks. The BAG coating had a uniform thickness of 35.38 ± 4.67 µm. The average surface roughness was significantly lower for BAG-coated implants, with less surface irregularities (3.34 ± 0.45 µm for uncoated implants, 1.45 ± 0.23 µm for BAG-coated implants). An adhesion strength of 18.51 ± 3.37 MPa was recorded for the BAG coating. After insertion into artificial bone, 66.23 ± 10.23% of the coating weight remained on the implant surface. A reduction in the thickness of the BAG coating only occurred in sites of high friction with bone after implant insertion into bovine bone. CONCLUSIONS: Coating titanium implants with 70S30C BAG nanoparticles is attainable through electrophoretic deposition and results in a homogenous coating layer with a moderately rough surface, considerable adhesion strength, and high coating stability during implant insertion. Int J Oral Maxillofac Implants 2023;38:591-606. doi: 10.11607/jomi.10272.

Nanoscale borosilicate bioactive glass for regenerative therapy of full-thickness skin defects in rabbit animal model.

Bioactive glass (BG) occupies a significant position in the field of hard and soft tissue regeneration. Different processing techniques and formulas have been introduced to expand their regenerative, angiogenic, and antibacterial properties. In the present study, a new formula of bborosilicate bioactive glass nanofibers was prepared and tested for its wound-healing efficacy in a rabbit animal model. The glass formula ((1-2) mol% of B2O3 (68-69) mol% of SiO2, and (29-30) mol% of CaO) was prepared primarily by the sol-gel technique followed by the electrospinning technique. The material was characterized for its ultrastructure using scanning electron microscopy, chemical composition using FTIR, and its dynamic in vitro biodegradability using ICP-AES. Twelve rabbits were subjected to surgical induction of full-thickness skin defects using a 1 cm2 custom-made stainlessteel skin punch. The bioactive glass nanofibers were used as a grafting material in 6 experimental rabbits, while the defects in the remaining rabbits were considered as the negative control samples. All defects were assessed clinically for the decrease in wound size and clinical signs of healing and histologically for angiogenesis, collagen density, inflammatory response, cell recruitment, epithelial lining, and appendages at 1,2 and 3 weeks following the intervention. Structural analysis of the glass fibers confirmed their nano-size which ranged from 150 to 700 nm. Moreover, the chemical analysis confirmed the presence of SiO2 and B2O3 groups within the structure of the nanofibers. Additionally, dynamic biodegradation analysis confirmed the rapid degradation of the material starting from the first 24 h and rapid leaching of calcium, silicon, and boron ions confirming its bioactivity. The wound healing study of the nanofibrous scaffold confirmed its ability to accelerate wound healing and the closure rate in healthy rabbits. Histological analysis of the defects confirmed the angiogenic, regenerative and antibacterial ability of the material throughout the study period. The results unveil the powerful therapeutic properties of the formed nanofibers and open a new gate for more experimental and clinical applications.

Biomimetic Coating of Titanium Surface Using Bioactive Glass Nanoparticles.

PURPOSE: The aim of this study was to coat titanium substrate with bioactive glass nanoparticles and characterize the deposited surface coat. MATERIALS AND METHODS: Amorphous bioglass nanoparticles < 20 nm in diameter were prepared using a modified sol-gel technique followed by a ball-milling process. The prepared nanoparticles were used to coat airborne particle-abraded titanium disks. The in vitro bioactivity of the bioglass nanopowder was confirmed using simulated body fluid. Coated surfaces were characterized in terms of microstructure, composition, thickness, phase structure, surface roughness, wettability, and tissue behavior in a rabbit model. RESULTS: Bioglass nanoparticles showed apatite formation under a scanning electron microscope (SEM) after 5 days, confirming that bioactivity was enhanced with increasing degradation rate for up to 2 weeks. An optimized deposition technique and heat-treatment process produced a homogenous coating with a uniform thickness of 32 to 39 µm. Chemical analysis confirmed the presence of silicon and calcium on the coated disks. Amorphous coated surfaces exhibited porous nano/microroughness with microcracks and super-hydrophilicity. The interface of the coated disks with subcutaneous tissue revealed good tissue adhesion, high cellular activity, and rich vascularization, with multinucleated cells in the microenvironment surrounding the coat, as confirmed using histomorphometric analysis. CONCLUSION: The results of this study show that it is feasible to coat titanium surfaces with bioactive glass nanoparticles with super-hydrophilicity and high biologic activity. These particles may promote the regenerative environment around dental implants.

Cultured keratinocytes on urinary bladder matrix scaffolds increase angiogenesis and help in rapid healing of wounds.

INTRODUCTION: Urinary Bladder Matrix (UBM) is an extracellular matrix (ECM) scaffold. It is now used in wound care management of partial and full-thickness wounds where conventional methods for wound care usually fail to give satisfactory results. OBJECTIVE: In this study, the authors are comparing the healing of full-thickness excisional wounds in New Zealand rabbits using either UBM scaffolds alone or in combination with cultured keratinocytes. The wounds were compared grossly and histologically. MATERIALS AND METHODS: It is a comparative controlled study including 40 full-thickness wounds in 2 groups. Group (A) wounds: treated with UBM scaffolds, Group (B) wounds: treated with UBM scaffolds with cultured keratinocytes. The wounds were examined grossly after 1, 2, and 3 weeks, and were examined histologically at the end of the 3rd week using ordinary hematoxylin-eosin staining techniques. RESULTS: All the wounds healed completely by the end of the 3rd week. Early wound contraction was significantly less in group B. More angiogenic response was evident in all specimens of group B. CONCLUSION: This study shows that adding cultured keratinocytes to the rough surface of the UBM scaffold may be beneficial in reducing early wound contraction and improving wound vascularity in treatment of full-thickness wounds.

Efficacy of Bioactive Glass Nanofibers Tested for Oral Mucosal Regeneration in Rabbits with Induced Diabetes.

The healing of oral lesions that are associated with diabetes mellitus is a matter of great concern. Bioactive glass is a highly recommended bioceramic scaffold for bone and soft tissue regeneration. In this study, we aimed to assess the efficacy of a novel formula of bioactive glass nanofibers in enhancing oral mucosal wound regeneration in diabetes mellitus. Bioactive glass nanofibres (BGnf) of composition (1-2) mol% of B2O3, (68-69) mol% of SiO2, and (29-30) mol% of CaO were synthesized via the low-temperature sol-gel technique followed by mixing with polymer solution, then electrospinning of the glass sol to produce nanofibers, which were then subjected to heat treatment. X-Ray Diffraction analysis of the prepared nanofibers confirmed its amorphous nature. Microstructure of BGnf simulated that of the fibrin clot with cross-linked nanofibers having a varying range of diameter (500-900 nm). The in-vitro degradation profile of BGnf confirmed its high dissolution rate, which proved the glass bioactivity. Following fibers preparation and characterization, 12 healthy New Zealand male rabbits were successfully subjected to type I diabetic induction using a single dose of intravenous injection of alloxan monohydrate. Two weeks after diabetes confirmation, the rabbits were randomly divided into two groups (control and experimental groups). Bilateral elliptical oral mucosal defects of 10 × 3.5 mm were created in the maxillary mucobuccal fold of both groups. The defects of the experimental group were grafted with BGnf, while the other group of defects considered as a control group. Clinical, histological, and immune-histochemical assessment of both groups of wounds were performed after one, two and three weeks' time interval. The results of the clinical evaluation of BGnf treated defects showed complete wound closure with the absence of inflammation signs starting from one week postoperative. Control defects, on the other hand, showed an open wound with suppurative exudate. On histological and immunohistochemical level, the BGnf treated defects revealed increasing in cell activity and vascularization with the absence of inflammation signs starting from one week time interval, while the control defects showed signs of suppurative inflammation at one week time interval with diminished vascularization. The results advocated the suitability of BGnf as bioscaffold to be used in a wet environment as the oral cavity that is full of microorganisms and also for an immune-compromised condition as diabetes mellitus.

Experimental formation of periodontal structure around titanium implants utilizing bone marrow mesenchymal stem cells: a pilot study.

Tissue engineering in the head and neck area, presents numerous advantages. One of the most remarkable advantages is that regeneration of only a small amount of tissue can be highly beneficial to the patient, particularly in the field of periodontal tissue regeneration. For decades, successful osseointegration has provided thousands of restorations that maintain normal function. With the increasing need to utilize dental implants for growing patients and enhance their function to simulate normal tooth physiology and proprioception, there appears to be an urgent need for t concept of periodontal tissue regeneration around dental implants. In the present work, 5 goats wer used for immediate implant placement post canine teeth extraction. Each goat received 2 implan fixtures; the control side received a porous hollow root-form poly (DL-Lactide-co-Glycolide) scaf around the titanium fixture, and the experimental side received the same scaffold but seeded with autogenous bone marrow-derived mesenchymal stem cells. One animal was killed 10 days postoperatively, and the others were killed after 1 month. The results showed that on th experimental side, periodontal-like tissue with newly formed bone was demonstrated both at 1 days and after 1 month, while the control specimens showed early signs of connective tissue regeneration around the titanium fixture at 10 days, but was not shown in the 1 month specimens. I can be concluded that undifferentiated mesenchymal stem cells were capable of differentiating t provide the 3 critical tissues required for periodontal tissue regeneration: cementum, bone, a periodontal ligament. This work may provide a new approach for periodontal tissue regeneration.

Regeneration of dentine/pulp-like tissue using a dental pulp stem cell/poly(lactic-co-glycolic) acid scaffold construct in New Zealand white rabbits.

With the expanding knowledge of tooth regeneration and biological mechanisms of functional dental tissue repair, current treatment strategies are beginning to give way to evolving fields such as tissue engineering and biomimetics. Dental pulp stem cells were isolated from rabbit teeth and seeded onto scaffolds prepared from 50/50 poly(lactic-co-glycolic acid) polymers using two different porogen particle sizes. These cell/scaffold constructs were then transplanted subcutaneously in the rabbits. The expanded rabbit dental pulp stem cells showed high proliferative and clonogenic capacities as well as the ability to give rise to mineralised-like tissues in vitro in culture flasks and after seeding them onto the scaffolds for 12 days. Histological evaluation of transplanted samples revealed the formation of osteodentine-like structures as well as tubular bilayered structures of vertically aligned parallel tubules resembling tubular-like dentine. Using a tissue engineering approach yielded tissues quite similar to normal dentine/pulp-like tissues that can perhaps be used later on for regenerative endodontic or operative procedures.

Dental Mesenchymal Stem Cell-Based Translational Regenerative Dentistry: From Artificial to Biological Replacement.

Dentistry is a continuously changing field that has witnessed much advancement in the past century. Prosthodontics is that branch of dentistry that deals with replacing missing teeth using either fixed or removable appliances in an attempt to simulate natural tooth function. Although such "replacement therapies" appear to be easy and economic they fall short of ever coming close to their natural counterparts. Complications that arise often lead to failures and frequent repairs of such devices which seldom allow true physiological function of dental and oral-maxillofacial tissues. Such factors can critically affect the quality of life of an individual. The market for dental implants is continuously growing with huge economic revenues. Unfortunately, such treatments are again associated with frequent problems such as peri-implantitis resulting in an eventual loss or replacement of implants. This is particularly influential for patients having co-morbid diseases such as diabetes or osteoporosis and in association with smoking and other conditions that undoubtedly affect the final treatment outcome. The advent of tissue engineering and regenerative medicine therapies along with the enormous strides taken in their associated interdisciplinary fields such as stem cell therapy, biomaterial development, and others may open arenas to enhancing tissue regeneration via designing and construction of patient-specific biological and/or biomimetic substitutes. This review will overview current strategies in regenerative dentistry while overviewing key roles of dental mesenchymal stem cells particularly those of the dental pulp, until paving the way to precision/translational regenerative medicine therapies for future clinical use.

Preservation and regeneration of alveolar bone by tissue-engineered implants.

Bone maintenance after dental extraction has a significant impact on the success of future treatment. The purpose of this study was to regenerate bone by implanting an engineered porous scaffold seeded with bone marrow mesenchymal stem cells (BMSCs) in a socket created by extraction of the lower left central incisor in rabbits, utilizing the principles of tissue engineering. It involved preparation and characterization of three-dimensional porous hollow root form scaffolds consisting of a poly-L-lactic acid:polyglycolic acid composite (PLG, 50:50), using a solvent casting/compression molding/particulate leaching technique. Porosity of the scaffolds was 83.71% with good interconnectivity and uniform distribution of the various pore sizes. The degraded scaffolds maintained their porosity and form for the first 2 weeks and their mass loss continued up to 6 weeks. The scaffolds developed viscoelastic behavior under dynamic compression; yet they lost their mechanical characteristics as they degraded. The scaffolds were seeded with BMSCs and examined by scanning electron microscopy. Cell proliferation and scaffold degradation were shown up to 2 weeks in vitro. The cultivated scaffolds were implanted in empty extraction sockets immediately after tooth removal. Four weeks later, bone regeneration was evaluated histologically in the healed sockets in three experimental groups: sockets left empty, sockets that received PLG without cells, and sockets that received PLG with cells. Radiographic evaluation, performed 4 weeks later for the three experimental groups, demonstrated preservation of alveolar bone walls in the extraction sockets that received PLG with cells as compared with the other two groups. The bone density profile for the healed sockets confirmed both histological and radiographic findings. The results of this study show promise in the area of dentoalveolar surgery, yet longitudinal studies under variable clinical situations would encourage the current application.

Axially vascularised mandibular constructs: Is it time for a clinical trial?

Applying regenerative therapies in the field of cranio-maxillofacial reconstruction has now become a daily practice. However, regeneration of challenging or irradiated bone defects following head and neck cancer is still far beyond clinical application. As the key factor for sound regeneration is the development of an adequate vascular supply for the construct, the current modalities using extrinsic vascularization are incapable of regenerating such complex defects. Our group has recently introduced the intrinsic axial vascularization technique to regenerate mandibular defects using the arteriovenous loop (AVL). The technique has shown promising results in terms of efficient vascularization and bone regeneration at the preclinical level. In this article, we have conducted a narrative literature review about using the AVL to vascularize tissue-engineering constructs at the preclinical level. We have also conducted a systematic literature review about applying the technique of axial vascularization in the field of craniofacial regeneration. The versatility of the technique and the possible challenges are discussed, and a suggested protocol for the first clinical trial applying the AVL technique for mandibular reconstruction is also presented.

Fabrication of polymer root form scaffolds to be utilized for alveolar bone regeneration.

Engineering dental tissues and organs is primarily motivated by a clinical need to restore these lost or diseased structures, in contrast to the use of harvested tissue. The present work focused on designing and characterizing scaffolds suitable for cultivation and implantation into the fresh extraction sockets of teeth, for the purpose of alveolar bone regeneration at a rate and quality higher than that of normal tissue healing for subsequent treatment with dental implants. Three-dimensional hollow root form scaffolds were prepared from poly-L-lactic acid/polyglycolic acid composites (50/50, 65/35, and 75/25 ratios), using the solvent casting compression molding particulate leaching technique. Two different salt particle sizes were used, 150-180 and 180-300 microm, to effect porogenesis. The scaffolds were characterized in vitro and in vivo. The highest percent porosity recorded was 75% with interconnectivity shown by scanning electron microscopy. The scaffolds demonstrated viscoelastic behavior and average strain in response to both static and dynamic forces that were suitable for them under bite-force magnitude anteriorly. The degradation of the root scaffolds depended on composite type, and on salt particle size. Tissue reaction favored samples made with large salt particle size.

Platelet rich plasma enhances osteoconductive properties of a hydroxyapatite-ß-tricalcium phosphate scaffold (Skelite) for late healing of critical size rabbit calvarial defects.

The use of platelet rich plasma (PRP) in bone repair remains highly controversial. In this work, we evaluated the effect of lyophilized PRP on bone regeneration when associated with a silicon stabilized hydroxyapatite tricalcium phosphate scaffold in a rabbit calvarial defect (Skelite). Critical defects were created in the calvaria of twenty-four rabbits. The periosteum was removed and the defects were either left empty or filled with allogeneic PRP gel; Skelite particles; Skelite and PRP gel. Four animals were killed after 4 weeks, 10 animals after 8 and 10 after 16 weeks. Specimens were processed for X-ray microtomography (µCT) and for resin embedded histology. µCT analysis revealed significant osteoid-like matrix and new bone deposition in PRP + Skelite group at both 8 and 16 weeks in respect to Skelite alone. Histologically, PRP + Skelite defects were highly cellular with more abundant osteoid deposition and more regular collagen fibres. Moreover, in vitro migration assays confirmed the chemotactic effect of PRP to endothelial and osteoprogenitor cells. We conclude that the addition of PRP influenced the local tissue microenvironment by providing key cryptic factors for regeneration, thereby enhancing progenitor cell recruitment, collagen and bone matrix deposition, and by creating a bridging interface between the scaffold and bone.

Nanoporosity significantly enhances the biological performance of engineered glass tissue scaffolds.

Nanoporosity is known to impact the performance of implants and scaffolds such as bioactive glass (BG) scaffolds, either by providing a higher concentration of bioactive chemical species from enhanced surface area, or due to inherent nanoscale topology, or both. To delineate the role of these two characteristics, BG scaffolds have been fabricated with nearly identical surface area (81 and 83±2 m(2)/g) but significantly different pore size (av. 3.7 and 17.7 nm) by varying both the sintering temperature and the ammonia concentration during the solvent exchange phase of the sol-gel fabrication process. In vitro tests performed with MC3T3-E1 preosteoblast cells on such scaffolds show that initial cell attachment is increased on samples with the smaller nanopore size, providing the first direct evidence of the influence of nanopore topography on cell response to a bioactive structure. Furthermore, in vivo animal tests in New Zealand rabbits (subcutaneous implantation) indicate that nanopores promote colonization and cell penetration into these scaffolds, further demonstrating the favorable effects of nanopores in tissue-engineering-relevant BG scaffolds.

Mandibular reconstruction using an axially vascularized tissue-engineered construct.

BACKGROUND: Current reconstructive techniques for continuity defects of the mandible include the use of free flaps, bone grafts, and alloplastic materials. New methods of regenerative medicine designed to restore tissues depend mainly on the so-called extrinsic neovascularization, where the neovascular bed originates from the periphery of the construct. This method is not applicable for large defects in irradiated fields. METHODS: We are introducing a new animal model for mandibular reconstruction using intrinsic axial vascularization by the Arterio-Venous (AV) loop. In order to test this model, we made cadaveric, mechanical loading, and surgical pilot studies on adult male goats. The cadaveric study aimed at defining the best vascular axis to be used in creating the AV loop in the mandibular region. Mechanical loading studies (3 points bending test) were done to ensure that the mechanical properties of the mandible were significantly affected by the designed defect, and to put a base line for further mechanical testing after bone regeneration. A pilot surgical study was done to ensure smooth operative and post operative procedures. RESULTS: The best vascular axis to reconstruct defects in the posterior half of the mandible is the facial artery (average length 32.5 ± 1.9 mm, caliber 2.5 mm), and facial vein (average length 33.3 ± 1.8 mm, caliber 2.6 mm). Defects in the anterior half require an additional venous graft. The defect was shown to be significantly affecting the mechanical properties of the mandible (P value 0.0204). The animal was able to feed on soft diet from the 3rd postoperative day and returned to normal diet within a week. The mandible did not break during the period of follow up (2 months). CONCLUSIONS: Our model introduces the concept of axial vascularization of mandibular constructs. This model can be used to assess bone regeneration for large bony defects in irradiated fields. This is the first study to introduce the concept of axial vascularization using the AV loop for angiogenesis in the mandibular region. Moreover, this is the first study aiming at axial vascularization of synthetic tissue engineering constructs at the site of the defect without any need for tissue transfer (in contrast to what was done previously in prefabricated flaps).

Alendronate PLGA microspheres with high loading efficiency for dental applications.

PURPOSE: Alendronate sodium, used systemically as a bone protective agent, proved to also be effective locally in various dental bone applications. Development of alendronate-loaded microspheres with high loading efficiency for such applications would be greatly challenged by the hydrophilicity and low MW of the drug. The aim of this study was to incorporate alendronate sodium, into poly (lactide-co-glycolide) (PLGA) microspheres (MS) with high loading efficiency. METHODS: Three multiple emulsion methods: water-in-oil-in-water (W/O/W), water-in-oil-in-oil (W/O(1)/O(2)) and solid-in-oil-in-oil (S/O(1)/O(2)) were tested. In addition to entrapment efficiency, MS were characterized for surface morphology, particle size, in vitro drug release and in vitro degradation of the polymer matrix. Alendronate microspheres with maximum drug loading and good overall in vitro performance were obtained using the W/O(1)/O(2) emulsion technique. RESULTS: Drug release from the microspheres exhibited a triphasic release pattern over a period of 13 days, the last fast release phase being associated with more rapid degradation of the PLGA matrix. CONCLUSIONS: Biocompatible, biodegradable PLGA microspheres incorporating alendronate sodium with high loading efficiency obtained in this study may offer promise as a delivery system for bisphosphonates in dental and probably other clinical applications.