Computed tomography and histological evaluation of xenogenic and biomimetic bone grafts in three-wall alveolar defects in minipigs

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.

     

Factors affecting the structure and properties of an injectable self-setting calcium phosphate foam

One of the main challenges in the investigation on calcium phosphate cements (CPC) lies in the introduction of macroporosity, without loosing the self-setting ability and injectability, characteristic of the cement-type materials. The benefits of macroporosity are related to the enhancement of bone regeneration mechanisms, such as angiogenesis and tissue ingrowth. In this work, the feasibility to obtain self-setting injectable macroporous hydroxyapatite foams by the incorporation of a protein-based foaming agent to a CPC is demonstrated. Albumen is combined with an alpha-tricalcium phosphate [Ca3(PO4)2, alpha-TCP] paste, which hydrolyzes to a calcium deficient hydroxyapatite during the setting reaction. A systematic study is presented, where the effect of different processing parameters is analyzed in terms of porosity, setting properties, injectability, and compressive strength. Self-setting foams with porosities up to 70%, which maintain their porous structure after injection, are obtained. These injectable foams can be used both for direct in vivo applications and for the fabrication of low temperature tissue engineering scaffolds.     

Calcium phosphate cements as drug delivery materials

Calcium phosphate cements are used as synthetic bone grafts, with several advantages, such as their osteoconductivity and injectability. Moreover, their low-temperature setting reaction and intrinsic porosity allow for the incorporation of drugs and active principles in the material. It is the aim of the present work to: a) provide an overview of the different approaches taken in the application of calcium phosphate cements for drug delivery in the skeletal system, and b) identify the most significant achievements. The drugs or active principles associated to calcium phosphate cements are classified in three groups, i) low molecular weight drugs; ii) high molecular weight biomolecules; and iii) ions.     

Novel magnesium phosphate cements with high early strength and antibacterial properties

Magnesium phosphate cements (MPCs) have been extensively used as fast setting repair cements in civil engineering. They have properties that are also relevant to biomedical applications, such as fast setting, early strength acquisition and adhesive properties. However, there are some aspects that should be improved before they can be used in the human body, namely their highly exothermic setting reaction and the release of potentially harmful ammonia or ammonium ions. In this paper a new family of MPCs was explored as candidate biomaterials for hard tissue applications. The cements were prepared by mixing magnesium oxide (MgO) with either sodium dihydrogen phosphate (NaH(2)PO(4)) or ammonium dihydrogen phosphate (NH(4)H(2)PO(4)), or an equimolar mixture of both. The exothermia and setting kinetics of the new cement formulations were tailored to comply with clinical requirements by adjusting the granularity of the phosphate salt and by using sodium borate as a retardant. The ammonium-containing MPC resulted in struvite (MgNH(4)PO(4)·6H(2)O) as the major reaction product, whereas the MPC prepared with sodium dihydrogen phosphate resulted in an amorphous product. Unreacted magnesium oxide was found in all the formulations. The MPCs studied showed early compressive strengths substantially higher than that of apatitic calcium phosphate cements. The Na-containing MPCs were shown to have antibacterial activity against Streptococcus sanguinis, which was attributed to the alkaline pH developed during the setting reaction.     

Relevance of microstructure for the early antibiotic release of fresh and pre-set calcium phosphate cements

Calcium phosphate cements (CPCs) have great potential as carriers for controlled release and vectoring of drugs in the skeletal system. However, a lot of work still has to be done in order to obtain reproducible and predictable release kinetics. A particular aspect that adds complexity to these materials is that they cannot be considered as stable matrices, since their microstructure evolves during the setting reaction. The aims of the present work were to analyze the effect of the microstructural evolution of the CPC during the setting reaction on the release kinetics of the antibiotic doxycycline hyclate and to assess the effect of the antibiotic on the microstructural development of the CPC. The incorporation of the drug in the CPC modified the textural and microstructural properties of the cements by acting as a nucleating agent for the heterogeneous precipitation of hydroxyapatite crystals, but did not affect its antibacterial activity. In vitro release experiments were carried out on readily prepared cements (fresh CPCs), and compared to those of pre-set CPCs. No burst release was found in any formulation. A marked difference in release kinetics was found at the initial stages; the evolving microstructure of fresh CPCs led to a two-step release. Initially, when the carrier was merely a suspension of α-TCP particles in water, a faster release was recorded, which rapidly evolved to a zero-order release. In contrast, pre-set CPCs released doxycycline following non-Fickian diffusion. The final release percentage was related to the total porosity and entrance pore size of each biomaterial.     

Biomimetic treatment on dental implants for short-term bone regeneration

Objectives

The main purpose of this work was to assess the short-term bone regenerative potential of new osteoconductive implants. The novelty of the study lies in the analysis of the effectiveness of a novel two-step treatment which combines shot-blasting with a thermo-chemical treatment, at very short times after implant placement in a minipig model.

Materials and methods

Three hundred twenty implants with four different surface treatments, namely bioactivated surfaces, micro-rough grit-blasted, micro-rough acid-etched and smooth as-machined titanium implants were placed into the bone of 20 minipigs. The percent of bone-to-implant contact was determined 3 days, 1, 2, 3 and 10 weeks after implant placement by histomorphometric analysis. Surface composition, topography and wettability of the implant specimens were analysed.

Results

The combination of shot-blasting and thermo-chemical treatment accelerated bone regeneration at early stages in comparison with all other treatments between day 3 and week 3 (p?<?0.05). The value of osseointegration attained at week 2 was maintained until the end of the experiment without any significant changes (percent direct contact?≈?85 %). This was mostly attributed to the ability of these implants to form in vivo a layer of apatitic mineral that coated the implant and could rapidly stimulate bone nucleation and growth from the implant surface.

Conclusions

The surface quality resulting from this treatment on cpTi provided dental implants with a unique ability of rapid bone regeneration and osseointegration.

Clinical relevance

This treatment represents a step forward in the direction of reducing the time prior to implant loading.     

Calcium phosphate cements: competitive drug carriers for the musculoskeletal system?

This paper attempts to provide an insight in the application of calcium phosphate cements (CPC) in the field of drug delivery devices for the musculoskeletal system. Their ability to set once implanted within the body, giving a highly microporous material, allows incorporation of many types of drugs and biologically active molecules, without losing activity and denaturalization. Additionally, by being injectable these materials can be used in the growing market for new technologies of minimally invasive surgery, and in the treatment of difficult accessible sites. All these characteristics, together with the excellent biological behaviour of CPC, make them good candidates for drug delivery devices to be used in the pharmacological treatment of a great number of diseases of the bone tissue.     

Micro-finite element models of bone tissue-engineering scaffolds

Tissue engineering is an emerging area in bioengineering at the frontiers between biomaterials, biology and biomechanics. The basic knowledge of the interactions between mechanical stimuli, cells and biomaterials is growing but the quantitative effect of mechanical stimuli on cells attached to biomaterials is still unknown. The objective of this study was to develop finite element models of various bone scaffolds based on calcium phosphate in order to calculate the load transfer from the biomaterial structure to the biological entities. Samples of porous calcium phosphate bone cement and biodegradable glass were scanned using micro-CT to determine the overall macroporosity, architecture and to develop finite element models of such materials. Compressive loads were applied on the models to simulate the in vitro environment of a bioreactor and stress and strain distributions were calculated. It was found that the effective Young's modulus was linearly related to the sample macroporosity. Results suggest that a 0.5% overall compressive strain can produce internal strain of the same order of magnitude as found in previous in vitro mechanically cell-strained studies or in mechanoregulation studies. Stress and strain concentrations due to the porous structures are possible candidate for favouring cell differentiation. Although strain distributions were similar between bone cement and porous glass, the stress distribution is clearly different. Future in vitro results could correlate the results obtained with such finite element study to explain the influence of mechanical stimuli on cell behaviour.     

Cellular response to calcium phosphate glasses with controlled solubility

In the last decades, the research on materials for bone regeneration has focused on materials that are degradable and capable of stimulating tissue regeneration. In this context, phosphate glasses offer an interesting alternative, given the wide range of solubility they present and their similarity with respect to the chemical composition of the bone mineral phase. In the current work, two different formulations of phosphate glasses in the system P(2)O(5)[bond]CaO[bond]Na(2)O[bond]TiO(2) are developed. The incorporation of TiO(2) into the glass network allows for better control of the glass dissolution rate. Although these glasses have been studied extensively from the physicochemical point of view, little is known about their biocompatibility. To evaluate the biological response to these materials, we have used a human skin fibroblast model. The cells were incubated in vitro following two different methods. The first was incubated in direct contact with the glasses and the second one, in the presence of their extracts. The effects of the materials on cell growth were determined by means of toxicity (WST assay), adhesion, and proliferation tests. The results showed that the in vitro behavior of soluble phosphate glasses is strongly affected by their solubility. On the other hand, the results showed that the cellular response is highly affected by the testing procedure.     

ADS-J1 Inhibits HIV-1 Entry by Interacting with gp120 and Does Not Block Fusion-Active gp41 Core Formation

We had shown that virus resistance to ADS-J1 was associated with amino acid changes in the envelope glycoprotein, mostly located in the gp120 coding region. Time-of-addition and endocytic virus transfer assays clearly demonstrated that ADS-J1 behaved as a gp120 inhibitor. ADS-J1-resistant virus was cross-resistant to the polyanion dextran sulfate, and recombination of gp120 recovered only the ADS-J1-resistant phenotype. In summary, ADS-J1 blocks an early step of virus entry that appears to be driven by gp120 alone.The essential steps of HIV-1 entry in the host cell offer several potential targets for the development of novel antiviral agents (19, 24, 33, 42). Agents that disrupt gp41-mediated membrane fusion, collectively called fusion inhibitors, were the first entry inhibitors to be approved for the treatment of HIV infection. Enfuvirtide (T20, Fuzeon) is a 36-amino-acid synthetic peptide with a sequence identical to a part of the C-terminal heptad repeat 2 (HR2) region of gp41 that binds to the N-terminal heptad repeat 1 (HR1) in an antiparallel manner, forming a coiled-coil structure during the prefusion step. Mutations in the highly conserved amino acid motif 36 to 45 in the HR1 domain confer resistance to T20 (35), providing strong evidence that HR1 is the site of interaction of T20. However, mutations in other regions of HIV-1 envelope (Env) have been also associated with T20 resistance (26, 27).Several low-molecular-weight (SMW) compounds have been identified as blockers of the initial steps of virus entry, including CCR5 coreceptor (33, 42). However, the identification of SMW compounds targeting gp41 has been elusive. A polyanionic compound, ADS-J1, was previously identified in silico as a potential candidate and shown to bind to gp41 peptides and interfere with the formation of the gp41 coiled-coil domain in an in vitro enzyme-linked immunosorbent assay (ELISA) model of HR1-HR2 interaction (16, 30, 31). Conversely, we had shown that ADS-J1 blocked the binding of HIV particles to lymphoid MT-4 cells and inhibited HIV replication at a time/site of interaction similar to those of the polyanion dextran sulfate (DS), a well-described, nonspecific inhibitor of virus entry (3). Moreover, at least four HIV-1 strains resistant to ADS-J1 were generated. The resistance to ADS-J1 was associated with gp120 based on the fact that the majority of the mutations were located in the gp120 coding sequence, mainly in the V3 loop region. Although three of the resistant strains contained mutations in gp41, one of them, HIV-1 ARA45C, did not (3). In addition, molecular modeling suggested that the gp120 V3 loop was the preferential binding site for ADS-J1 onto HIV-1, and mutations induced by the inhibitor significantly changed the stereoelectronic properties of the gp120 surface, justifying a marked drop in the affinity of ADS-J1 toward an ADS-J1-resistant HIV-1 strain (36). At that time, we considered conclusive the evidence of the mode of action of ADS-J1.More recently, Wang et al. (43) suggested that ADS-J1 could bind directly to a trimeric peptide containing the gp41 pocket region (IQN17) in a surface plasmon resonance (SPR) assay and indicated that ADS-J1 can be used as a lead compound for the design of novel HIV-1 fusion inhibitors (44). Therefore, we thought it relevant to provide further evidence of the mode of action of ADS-J1.