ABSTRACT
One of the most widely used materials for bone graft substitution is ß-Tricalcium phosphate (ß-TCP; ß-Ca3(PO4)2). ß-TCP is typically produced by sintering in air or vacuum. During this process, evaporation of phosphorus (P) species occurs, leading to the formation of a calcium-rich alkaline layer. It was recently shown that the evaporation of P species could be prevented by co-sintering ß-TCP with dicalcium phosphate (DCPA; CaHPO4; mineral name: monetite). The aim of this study was to see how a change of sintering atmosphere could affect the physico-chemical and biological properties of ß-TCP. For this purpose, three experimental groups were considered: ß-TCP cylinders sintered in air and subsequently polished to remove the surface layer (control group); the same polished cylinders after subsequent annealing at 500 °C in air to generate a calcium-rich alkaline layer (annealed group); and finally, ß-TCP cylinders sintered in a monetite-rich atmosphere and subsequently polished (monetite group). XPS analysis confirmed that cylinders from the annealed group had a significantly higher Ca/P molar ratio at their surface than that of the control group while this ratio was significantly lower for the cylinders from the monetite group. Sintering ß-TCP in the monetite-rich atmosphere significantly reduced the grain size and increased the density. Changes of surface composition affected the activity of osteoclasts seeded onto the surfaces, since annealed ß-TCP cylinders were significantly less resorbed than ß-TCP cylinders sintered in the monetite-rich atmosphere. This suggests that an increase of the surface Ca/P molar ratio leads to a decrease of osteoclastic resorption. STATEMENT OF SIGNIFICANCE: Minimal changes of surface and bulk (< 1%) composition have major effects on the ability of osteoclasts to resorb ß-tricalcium phosphate (ß-TCP), one of the most widely used ceramics for bone substitution. The results presented in this study are thus important for the calcium phosphate community because (i) ß-TCP may have up to 5% impurities according to ISO and ASTM standards and still be considered to be "pure ß-TCP", (ii) ß-TCP surface properties are generally not considered during biocompatibility assessment and (iii) a rationale can be proposed to explain the various inconsistencies reported in the literature on the biological properties of ß-TCP.
Subject(s)
Bone Resorption , Calcium , Humans , Calcium Phosphates/pharmacology , AtmosphereABSTRACT
Heterotopic ossification (HO) is a condition triggered by an injury leading to the formation of mature lamellar bone in extraskeletal soft tissues. Despite being a frequent complication of orthopedic and trauma surgery, brain and spinal injury, the etiology of HO is poorly understood. The aim of this study is to evaluate the hypothesis that a sustained local ionic homeostatic imbalance (SLIHI) created by mineral formation during tissue calcification modulates inflammation to trigger HO. This evaluation also considers the role SLIHI could play for the design of cell-free, drug-free osteoinductive bone graft substitutes. The evaluation contains five main sections. The first section defines relevant concepts in the context of HO and provides a summary of proposed causes of HO. The second section starts with a detailed analysis of the occurrence and involvement of calcification in HO. It is followed by an explanation of the causes of calcification and its consequences. This allows to speculate on the potential chemical modulators of inflammation and triggers of HO. The end of this second section is devoted to in vitro mineralization tests used to predict the ectopic potential of materials. The third section reviews the biological cascade of events occurring during pathological and material-induced HO, and attempts to propose a quantitative timeline of HO formation. The fourth section looks at potential ways to control HO formation, either acting on SLIHI or on inflammation. Chemical, physical, and drug-based approaches are considered. Finally, the evaluation finishes with a critical assessment of the definition of osteoinduction. STATEMENT OF SIGNIFICANCE: The ability to regenerate bone in a spatially controlled and reproducible manner is an essential prerequisite for the treatment of large bone defects. As such, understanding the mechanism leading to heterotopic ossification (HO), a condition triggered by an injury leading to the formation of mature lamellar bone in extraskeletal soft tissues, would be very useful. Unfortunately, the mechanism(s) behind HO is(are) poorly understood. The present study reviews the literature on HO and based on it, proposes that HO can be caused by a combination of inflammation and calcification. This mechanism helps to better understand current strategies to prevent and treat HO. It also shows new opportunities to improve the treatment of bone defects in orthopedic and dental procedures.
Subject(s)
Bone Substitutes , Calcinosis , Ossification, Heterotopic , Bone and Bones , Calcinosis/complications , Humans , Inflammation , Ossification, Heterotopic/etiologyABSTRACT
Some synthetic bone graft substitutes (BGS) can trigger ectopic bone formation, which is the hallmark of osteoinduction and the most important prerequisite for the repair of large bone defects. Unfortunately, measuring or predicting BGS osteoinductive potential based on in vitro experiments is currently impossible. A recent study claimed that synthetic BGS can induce bone formation ectopically if they create a local homeostatic imbalance during their in vivo mineralization. This raised the hope that a simple cell free in vitro mineralization experiment would correlate with osteoinduction. The aim of the present study was therefore to assess the ability of a quantitative in vitro mineralization test to predict and rank the osteoinductive potential of BGS. Eight calcium phosphate BGS already tested ectopically in 9 different in vivo studies were used for that purpose. The experiment was able to identify materials that are reliably osteoinductive from those that are not, but was inaccurate in ranking the osteoinductive materials between each other. Chemical contaminants (Ca2+, Mg2+, H+, OH-, PO43-) present in some of the BGS affected the in vitro mineralization experiment results, but not in a direction that could explain the different rankings. In conclusion, this study suggests that an in vitro experiment can be used as a fast and reliable screening tool to identify osteoinductive BGS and underline the need to study ionic contaminants on calcium phosphate BGS.
Subject(s)
Bone Substitutes , Calcium Phosphates/pharmacology , Microscopy, Electron, Scanning , OsteogenesisABSTRACT
OBJECTIVES: This study aimed to compare the performance of a xenograft (XG) and a biomimetic synthetic graft (SG) in three-wall alveolar defects in minipigs by means of 3D computerised tomography and histology. MATERIALS AND METHODS: Eight minipigs were used. A total of eight defects were created in the jaw of each animal, three of which were grafted with XGs, three with SGs, and two were left empty as a negative control. The allocation of the different grafts was randomised. Four animals were euthanised at 6 weeks and four at 12 weeks. The grafted volume was then measured by spiral computed tomography to assess volume preservation. Additionally, a histological analysis was performed in undecalcified samples by backscattered scanning electron microscopy and optical microscopy after Masson's trichrome staining. RESULTS: A linear mixed-effects model was applied considering four fixed factors (bone graft type, regeneration time, anatomic position, and maxilla/mandible) and one random factor (animal). The SG exhibited significantly larger grafted volume (19%) than the XG. The anterior sites preserved better the grafted volume than the posterior ones. Finally, regeneration time had a positive effect on the grafted volume. Histological observations revealed excellent osseointegration and osteoconductive properties for both biomaterials. Some concavities found in the spheroidal morphologies of SGs were associated with osteoclastic resorption. CONCLUSIONS: Both biomaterials met the requirements for bone grafting, i.e. biocompatibility, osseointegration, and osteoconduction. Granule morphology was identified as an important factor to ensure a good volume preservation. CLINICAL RELEVANCE: Whereas both biomaterials showed excellent osteoconduction, SGs resulted in better volume preservation.
Subject(s)
Biomimetics , Bone Transplantation , Animals , Bone Regeneration , Mandible , Swine , Swine, Miniature , TomographyABSTRACT
OBJECTIVE: To describe a novel surgical approach to treat a critical-sized bone defect due to severe, radial atrophic nonunion in a miniature dog. STUDY DESIGN: Case report ANIMAL: A 1-year-old Yorkshire terrier with a critical-sized left radial defect after failed internal fixation of a transverse radial fracture. METHODS: Computed tomographic (CT) images of the radius were imported for three-dimensional (3D) printing of a custom-designed synthetic 3D-printed ß-tricalcium phosphate (ß-TCP) scaffold. The radius was exposed, and the ß-TCP scaffold was press-fitted in the bone gap underneath the plate. Recombinant human bone morphogenic protein-2 (RhBMP-2) collagen sponges were squeezed to soak the scaffold with growth factor and then placed on both sides of the synthetic graft. Two additional cortical screws were also placed prior to routine closure of the surgical site. RESULTS: Radiographic examination was consistent with complete healing of the radius defect 4 months after surgery. The bone plate was removed 10 months after surgery. According to CT examination 18 months after surgery, there was no evidence of the synthetic graft; instead, complete corticalization of the affected area was noted. Complete functional recovery was observed until the last clinical follow-up 36 months postoperatively. CONCLUSION: Screw fixation and use of a 3D-printed ceramic scaffold augmented with rhBMP-2 resulted in excellent bone regeneration of the nonunion and full recovery of a miniature breed dog. CLINICAL SIGNIFICANCE: The therapeutic approach used in this dog could be considered as an option for treatment of large-bone defects in veterinary orthopedics, especially for defects affecting the distal radius of miniature dogs.
Subject(s)
Bone Morphogenetic Protein 2/metabolism , Bone Transplantation/veterinary , Calcium Phosphates/chemistry , Dogs/surgery , Fractures, Malunited/veterinary , Printing, Three-Dimensional , Radius Fractures/veterinary , Transforming Growth Factor beta/metabolism , Animals , Bone Transplantation/instrumentation , Dogs/injuries , Fractures, Malunited/surgery , Fractures, Malunited/therapy , Male , Radius Fractures/surgery , Radius Fractures/therapy , Recombinant Proteins/metabolismABSTRACT
Although autografts are considered to be the gold standard treatment for reconstruction of large bone defects resulting from trauma or diseases, donor site morbidity and limited availability restrict their use. Successful bone repair also depends on sufficient vascularization and to address this challenge, novel strategies focus on the development of vascularized biomaterial scaffolds. This pilot study aimed to investigate the feasibility of regenerating large bone defects in sheep using 3D-printed customized calcium phosphate scaffolds with or without surgical vascularization. Pre-operative computed tomography scans were performed to visualize the metatarsus and vasculature and to fabricate customized scaffolds and surgical guides by 3D printing. Critical-sized segmental defects created in the mid-diaphyseal region of the metatarsus were either left empty or treated with the 3D scaffold alone or in combination with an axial vascular pedicle. Bone regeneration was evaluated 1, 2 and 3 months post-implantation. After 3 months, the untreated defect remained non-bridged while the 3D scaffold guided bone regeneration. The presence of the vascular pedicle further enhanced bone formation. Histology confirmed bone growth inside the porous 3D scaffolds with or without vascular pedicle inclusion. Taken together, this pilot study demonstrated the feasibility of precised pre-surgical planning and reconstruction of large bone defects with 3D-printed personalized scaffolds.
Subject(s)
Calcium Phosphates/chemistry , Metatarsus/surgery , Tissue Scaffolds/chemistry , Animals , Biocompatible Materials/chemistry , Bone Regeneration/physiology , Pilot Projects , Printing, Three-Dimensional , SheepABSTRACT
Several mechanisms proposed to explain the osteoinductive potential of calcium phosphates involve surface mineralization ("bioactivity") and mention the occurrence of concentration gradients between the inner and the outer part of the implanted material. Determining the evolution of the local chemical environment occurring inside the pores of an implanted bone graft substitute (BGS) is therefore highly relevant. A quantitative and fast method was developed to measure the chemical changes occurring within the pores of ß-Tricalcium Phosphate (ß-TCP) granules incubated in a simulated body fluid. A factorial design of experiment was used to test the effect of particle size, specific surface area, microporosity, and purity of the ß-TCP granules. Large pH, calcium and phosphate concentration changes were observed inside the BGS and lasted for several days. The kinetics and magnitude of these changes (up to 2 pH units) largely depended on the processing and properties of the granules. Interestingly, processing parameters that increased the kinetics and magnitude of the local chemical changes are parameters considered to favor calcium phosphate osteoinduction, suggesting that the model might be useful to predict the osteoinductive potential of BGSs. STATEMENT OF SIGNIFICANCE: Recent results suggest that in situ mineralization of biomaterials (polymers, ceramics, metals) might be key in their ability to trigger ectopic bone formation. This is the reason why the effect on in situ mineralization of various synthesis parameters of ß-tricalcium phosphate granules was studied (size, microporosity, specific surface area, and Ca/P molar ratio). To the best of our knowledge, this is the first article devoted to the chemical changes occurring within the pores of a bone graft substitute. We believe that the manuscript will prove to be highly important in the design and mechanistic understanding of drug-free osteoinductive biomaterials.
Subject(s)
Bone Substitutes/chemistry , Calcium Phosphates/chemistry , Particle Size , Porosity , Prostheses and Implants , SolubilityABSTRACT
IMPACT STATEMENT: This work reports a new bone substitute made of precipitated apatite crystals that resemble in composition and crystallinity to the mineral phase of bone. The bone regeneration capacity of this synthetic biomimetic calcium phosphate (SBCP) was studied by using an original model of vertical bone regeneration with cups on the calvaria of rats. After 4 weeks, a significantly higher bone growth was found with SBCP compared with deproteinized bovine bone matrix and empty controls. This rapid vertical bone regeneration indicated that this new biomaterial is particularly interesting for filling bone defects in oral surgery.
Subject(s)
Biomimetics , Bone Regeneration/drug effects , Bone Substitutes/pharmacology , Calcium Phosphates/pharmacology , Skull/cytology , Animals , Male , Rats , Rats, Wistar , Skull/drug effects , Skull/physiologyABSTRACT
There is an urgent need of synthetic bone grafts with enhanced osteogenic capacity. This can be achieved by combining biomaterials with exogenous growth factors, which however can have numerous undesired side effects, but also by tuning the intrinsic biomaterial properties. In a previous study, we showed the synergistic effect of nanostructure and pore architecture of biomimetic calcium deficient hydroxyapatite (CDHA) scaffolds in enhancing osteoinduction, i.e. fostering the differentiation of mesenchymal stem cells to bone forming cells. This was demonstrated by assessing bone formation after implanting the scaffolds intramuscularly. The present study goes one step forward, since it analyzes the effect of the geometrical features of the same CDHA scaffolds, obtained either by 3D-printing or by foaming, on the osteogenic potential and resorption behaviour in a bony environment. After 6 and 12â¯weeks of intraosseous implantation, both bone formation and material degradation had been drastically affected by the macropore architecture of the scaffolds. Whereas nanostructured CDHA was shown to be highly osteoconductive both in the robocast and foamed scaffolds, a superior osteogenic capacity was observed in the foamed scaffolds, which was associated with their higher intrinsic osteoinductive potential. Moreover, they showed a significantly higher cell-mediated degradation than the robocast constructs, with a simultaneous and progressive replacement of the scaffold by new bone. In conclusion, these results demonstrate that the control of macropore architecture is a crucial parameter in the design of synthetic bone grafts, which allows fostering both material degradation and new bone formation. Statement of Significance 3D-printing technologies open new perspectives for the design of patient-specific bone grafts, since they allow customizing the external shape together with the internal architecture of implants. In this respect, it is important to design the appropriate pore geometry to maximize the bone healing capacity of these implants. The present study analyses the effect of pore architecture of nanostructured hydroxyapatite scaffolds, obtained either by 3D-printing or foaming, on the osteogenic potential and scaffold resorption in an in vivo model. While nanostructured hydroxyapatite showed excellent osteoconductive properties irrespective of pore geometry, we demonstrated that the spherical, concave macropores of foamed scaffolds significantly promoted both material resorption and bone regeneration compared to the 3D-printed scaffolds with orthogonal-patterned struts and therefore prismatic, convex macropores.
Subject(s)
Calcium Phosphates/chemistry , Nanostructures/chemistry , Osteogenesis , Printing, Three-Dimensional , Tissue Scaffolds/chemistry , Animals , Dogs , Durapatite/chemistry , Imaging, Three-Dimensional , Nanostructures/ultrastructure , Porosity , X-Ray MicrotomographyABSTRACT
Calcium phosphates have long been used as synthetic bone grafts. Recent studies have shown that the modulation of composition and textural properties, such as nano-, micro- and macro-porosity, is a powerful strategy to control and synchronize material resorption and bone formation.Biomimetic calcium phosphates, which closely mimic the composition and structure of bone mineral, can be produced using low-temperature processing routes, and offer the possibility to modulate the material properties to a larger extent than conventional high temperature sintering processes.Advanced technologies open up new possibilities in the design of bioceramics for bone regeneration; 3D-printing technologies, in combination with the development of hybrid materials with enhanced mechanical properties, supported by finite element modelling tools, are expected to enable the design and fabrication of mechanically competent patient-specific bone grafts.The association of ions, drugs and cells allows leveraging of the osteogenic potential of bioceramic scaffolds in compromised clinical situations, where the intrinsic bone regeneration potential is impaired. Cite this article: EFORT Open Rev 2018;3 DOI: 10.1302/2058-5241.3.170056.
ABSTRACT
Direct ink writing (DIW) techniques open up new possibilities for the fabrication of patient-specific bone grafts. Self-setting calcium phosphate inks, which harden at low temperature, allow obtaining nanostructured scaffolds with biomimetic properties and enhanced bioactivity. However, the slow hardening kinetics hampers the translation to the clinics. Different hydrothermal treatments for the consolidation of DIW scaffolds fabricated with an α-tricalcium phosphate /pluronic F127 ink were explored, comparing them with a biomimetic treatment. Three different scaffold architectures were analysed. The hardening process, associated to the conversion of α-tricalcium phosphate to hydroxyapatite was drastically accelerated by the hydrothermal treatments, reducing the time for complete reaction from 7â¯days to 30 minutes, while preserving the scaffold architectural integrity and retaining the nanostructured features. ß-tricalcium phosphate was formed as a secondary phase, and a change of morphology from plate-like to needle-like crystals in the hydroxyapatite phase was observed. The binder was largely released during the treatment. The hydrothermal treatment resulted in a 30% reduction of the compressive strength, associated to the residual presence of ß-tricalcium phosphate. Biomimetic and hydrothermally treated scaffolds supported the adhesion and proliferation of rat mesenchymal stem cells, indicating a good suitability for bone tissue engineering applications. STATEMENT OF SIGNIFICANCE: 3D plotting has opened up new perspectives in the bone regeneration field allowing the customisation of synthetic bone grafts able to fit patient-specific bone defects. Moreover, this technique allows the control of the scaffolds' architecture and porosity. The present work introduces a new method to harden biomimetic hydroxyapatite 3D-plotted scaffolds which avoids high-temperature sintering. It has two main advantages: i) it is fast and simple, reducing the whole fabrication process from the several days required for the biomimetic processing to a few hours; and ii) it retains the nanostructured character of biomimetic hydroxyapatite and allows controlling the porosity from the nano- to the macroscale. Moreover, the good in vitro cytocompatibility results support its suitability for cell-based bone regeneration therapies.
Subject(s)
Calcium Phosphates/chemistry , Ink , Mesenchymal Stem Cells/metabolism , Nanostructures/chemistry , Polyethylenes/chemistry , Polypropylenes/chemistry , Tissue Scaffolds/chemistry , Animals , Cell Adhesion , Compressive Strength , Hot Temperature , Mesenchymal Stem Cells/cytology , Rats , Rats, Inbred LewABSTRACT
Some biomaterials are osteoinductive, that is, they are able to trigger the osteogenic process by inducing the differentiation of mesenchymal stem cells to the osteogenic lineage. Although the underlying mechanism is still unclear, microporosity and specific surface area (SSA) have been identified as critical factors in material-associated osteoinduction. However, only sintered ceramics, which have a limited range of porosities and SSA, have been analyzed so far. In this work, we were able to extend these ranges to the nanoscale, through the foaming and 3D-printing of biomimetic calcium phosphates, thereby obtaining scaffolds with controlled micro- and nanoporosity and with tailored macropore architectures. Calcium-deficient hydroxyapatite (CDHA) scaffolds were evaluated after 6 and 12 weeks in an ectopic-implantation canine model and compared with two sintered ceramics, biphasic calcium phosphate and ß-tricalcium phosphate. Only foams with spherical, concave macropores and not 3D-printed scaffolds with convex, prismatic macropores induced significant ectopic bone formation. Among them, biomimetic nanostructured CDHA produced the highest incidence of ectopic bone and accelerated bone formation when compared with conventional microstructured sintered calcium phosphates with the same macropore architecture. Moreover, they exhibited different bone formation patterns; in CDHA foams, the new ectopic bone progressively replaced the scaffold, whereas in sintered biphasic calcium phosphate scaffolds, bone was deposited on the surface of the material, progressively filling the pore space. In conclusion, this study demonstrates that the high reactivity of nanostructured biomimetic CDHA combined with a spherical, concave macroporosity allows the pushing of the osteoinduction potential beyond the limits of microstructured calcium phosphate ceramics.
ABSTRACT
Although calcium phosphate cements (CPCs) are used for bone regeneration in a wide range of clinical applications, various physicochemical phenomena are known to hinder their potential use in minimally invasive surgery or in highly vascularized surgical sites, mainly because of their lack of injectability or their low washout resistance. The present work shows that the combination of CPCs with an inverse-thermoresponsive hydrogel is a good strategy for finely tuning the cohesive and rheological properties of CPCs to achieve clinical acceptable injectability to prevent phase separation during implantation and cohesion to avoid washout of the paste. The thermoresponsive CPC developed combines alpha-tricalcium phosphate with an aqueous solution of pluronic F127, which exhibits an inverse thermoresponsive behaviour, with a gelling transformation at around body temperature. These novel CPCs exhibited temperature-dependent properties. Addition of the polymer enhanced the injectability of the paste, even at a low liquid-to-powder ratio, and allowed the rheological properties of the cement to be tuned, with the injection force decreasing with the temperature of the paste. Moreover, the cohesion of the paste was also temperature-dependent and increased as the temperature of the host medium increased due to gelling induced in the paste. The thermoresponsive cement exhibited excellent cohesion and clinically acceptable setting times at 37°C, irrespective of the initial temperature of the paste. The addition of pluronic F127 slightly delayed the setting reaction in the early stages but did not hinder the full transformation to calcium-deficient hydroxyapatite. Moreover, the frozen storage of premixed thermoresponsive cement pastes was explored, the main physicochemical properties of the cements being maintained upon thawing, even after 18months of frozen storage. This avoids the need to mix the cement in the operating theatre and allows its use off-the-shelf. The reverse thermoresponsive cements studied herein open up new perspectives in the surgical field, where the sequential gelling/hardening of these novel cements could allow for a better and safer clinical application. STATEMENT OF SIGNIFICANCE: Calcium phosphate cements are attractive bone substitutes due to their similarity to the bone mineral phase. Although they can be injectable, cohesion and stability of the paste are crucial in terms of performance and safety. A common strategy is the combination with hydrogels. However, this often results in a decrease of viscosity with increasing temperature, which can lead to extravasation and particle leakage from the bone defect. The preferred evolution would be the opposite: a low viscosity would enhance mixing and injection, and an instantaneous increase of viscosity after injection would ensure washout resistance to the blood flow. Here we develop for the first time a calcium phosphate cement exhibiting reverse thermoresponsive properties using a poloxamer featuring inverse thermal gelling.
Subject(s)
Calcium Phosphates/chemistry , Poloxamer/chemistry , Temperature , Bone Cements/chemistry , Injections , Microscopy, Electron, Scanning , Optical Imaging , Time Factors , X-Ray DiffractionABSTRACT
The porosity of a calcium phosphate cement is a key parameter as it affects several important properties of the cement. However, a successful, non-destructive porosity measurement method that does not include drying has not yet been reported for calcium phosphate cements. The aim of this study was to evaluate isopropanol solvent exchange as such a method. Two different types of calcium phosphate cements were used, one basic (hydroxyapatite) and one acidic (brushite). The cements were allowed to set in an aqueous environment and then immersed in isopropanol and stored under three different conditions: at room temperature, at room temperature under vacuum (300 mbar) or at 37â. The specimen mass was monitored regularly. Solvent exchange took much longer time to reach steady state in hydroxyapatite cements compared to brushite cements, 350 and 18 h, respectively. Furthermore, the immersion affected the quasi-static compressive strength of the hydroxyapatite cements. However, the strength and phase composition of the brushite cements were not affected by isopropanol immersion, suggesting that isopropanol solvent exchange can be used for brushite calcium phosphate cements. The main advantages with this method are that it is non-destructive, fast, easy and the porosity can be evaluated while the cements remain wet, allowing for further analysis on the same specimen.
Subject(s)
Bone Cements/chemistry , Calcium Phosphates/chemistry , Hydroxyapatites/chemistry , Compressive Strength , Materials Testing , PorosityABSTRACT
Determining the contact angle of a liquid on a solid surface is a simple method to assess the surface wettability. The most common method to measure the contact angle of a liquid consists of capturing the profile of a sessile drop of a few microliters on the surface using an optical system. Currently, this is a widely used technique to analyze wettability both in researched materials and in products of multiple technological fields. However, the drop dispensed by a traditional macroscopic contact angle meter is too big to assess the wettability properties of individual topographical features and/or chemical patterns at the micro/nanoscale. Recently, contact angle meters that can discharge drops that are microscopic, with volumes in the range of 1 × 10(-3) to 10(-5) µL have been developed. The novel microscopic contact angle meter uses a pneumatic injection system to discharge the drop of the liquid through a capillary of a few micrometers of internal diameter and a high-resolution ultrafast digital camera. We have tested different biosurfaces - microimprinted polymers for biosensors, calcium-phosphate cements with different topographical microfeatures, orthodontic wires - and assessed the potential applicability in the field in comparison with the conventional macroscopic contact angle meters. This protocol describes the basic tasks needed to test wettability on biosurfaces with a microscopic contact angle meter. The focus of the protocol is on the challenging methodological steps and those that differentiate the use of this equipment to the use of a traditional macroscopic contact angle meter.