Polymer-coated bioactive glass S53P4 increases VEGF and TNF expression in an induced membrane model in vivo

The two-stage induced-membrane technique for treatment of large bone defects has become popular among orthopedic surgeons. In the first operation, the bone defect is filled with poly(methyl methacrylate) (PMMA), which is intended to produce a membrane around the implant. In the second operation, PMMA is replaced with autograft or allograft bone. Bioactive glasses (BAGs) are bone substitutes with bone-stimulating and angiogenetic properties. The aim of our study was to evaluate the inductive vascular capacity of BAG-S53P4 and poly(lactide-co-glycolide) (PLGA)-coated BAG-S53P4 for potential use as bone substitutes in a single-stage induced-membrane technique. Sintered porous rods of BAG-S53P4, PLGA-coated BAG-S53P4 and PMMA were implanted in the femur of 36 rabbits for 2, 4 and 8 weeks. The expression of vascular endothelial growth factor (VEGF) and tumor necrosis factor alpha (TNF) in the induced membranes of implanted materials was analyzed with real-time quantitative polymerase chain reaction and compared with histology. Both uncoated BAG-S53P4 and PLGA-coated BAG-S53P4 increase expression of VEGF and TNF, resulting in higher amounts of capillary beds, compared with the lower expression of VEGF and less capillary beads observed for negative control and PMMA samples. A significantly higher expression of VEGF was observed for PLGA-coated BAG-S53P4 than for PMMA at 8 weeks (p <0.036). VEGF and TNF expression in the induced membrane of BAG-S53P4 and PLGA-coated BAG-S53P4 is equal or superior to PMMA, the "gold standard" material used in the induced-membrane technique. Furthermore, the VEGF and TNF expression for PLGA-coated BAG-S53P4 increased during follow-up.Peer reviewe

Bioactive glass S53P4 scaffolds : a preclinical study of S53P4 scaffolds and their potential use as a bone graft substitute in an induced membrane technique

Background and aim The treatment of bone defects is challenging regardless of the aetiology of the defect. Traditionally, long-bone defects are treated with autologous bone grafting, vascular bone grafting, distraction osteogenesis, or with tumour prosthesis techniques. New treatment modalities are emerging and the induced membrane technique (Masquelet technique, IMT) has been presented to treat large long-bone defects. The IMT is a two-staged surgical procedure where the induced membrane is regarded as the key element. In the first stage, the bone defect area is carefully debrided, supplemented with proper soft-tissue reconstruction, and filled with a polymethylmetacrylate (PMMA) spacer. This PMMA spacer induces the formation of a foreign body membrane around the bone defect, i.e. an induced membrane (IM). The IM conveys neovascularization to the bone defect site, isolates the bone defect site hindering bone graft resorption, serves as a source of mesenchymal stem cells (MSC), and provides an osteogenic stimulative effect via secretion of bone morphogenic proteins (BMP). In the second stage, typically 4 to 8 weeks after the initial surgery, the IM is carefully incised, the PMMA spacer removed, and the defect filled with autologous bone graft. To replace the two-staged procedure and to eliminate the need for autologous bone grafting, we developed a bioactive glass (BAG) scaffold by sintering BAG-S53P4 granules for use in a single-stage IMT without the need for autologous bone graft. Furthermore, selected scaffolds were coated with poly(DL-lactide-co-glycolide) (PLGA) to increase the mechanical properties of the scaffold and to potentially induce a more potent IM compared to BAG-S53P4 alone. We sought to introduce a BAG scaffold that can induce a membrane similar to PMMA and that allows bone formation at the bone defect site in the metaphyseal area of a long bone. Materials and methods The present study consists of in vitro and in vivo evaluations of BAG-S53P4(±PLGA) scaffolds. The in vitro study was conducted on macrophages and MSCs to evaluate the immunomodulatory and osteogenic effects of BAG scaffolds. The in vivo study was conducted on New Zealand White (NZW) rabbits to assess IM formation and bone formation in a bone defect model located in the metaphysis of a long bone. Results The BAG-S53P4 scaffolds demonstrated a clear immunomodulatory and osteogenic effect on macrophages and MSCs, respectively, and this effect was attenuated by the PLGA coating. The in vitro results of osteogenic ability were similar as those from the in vivo experiments in rabbits. New bone formation was observed in BAG-S53P4(±PLGA) scaffolds at the 2-week timepoint, and bone formation increased during the 8-week follow up. The IMs of PMMA and BAG-S53P4(±PLGA) had a similar appearance in microscopic evaluations throughout follow up. The aforementioned IMs were less fibrotic and displayed more abundant capillary formation than control samples during follow up. The highest number of capillaries at the 8-week timepoint was observed in BAG-S53P4-PLGA IMs. The IMs of BAG-S53P4(±PLGA) showed similar or superior mRNA expression rates when compared with PMMA IMs regarding tumour necrosis factor alpha (TNFα), vascular endothelial growth factor (VEGF), and BMP-2, -4, and -7. Conclusion In this study, the sintered BAG-S53P4(±PLGA) scaffold addresses the key requirements of successful bone regeneration in the metaphyseal area of long bones, namely sintered BAG-S53P4(±PLGA) scaffolds are osteoconductive; induces a membrane that serves as a source of inflammatory mediators and growth factors; has the capacity to stimulate MSCs towards osteogenic differentiation (osteostimulation); and immunomodulates inflammatory cells that can possibly be tailored for a specific clinical need. Thus, BAG-S53P4(±PLGA) scaffolds demonstrate the requisite properties for potential use as a bone graft substitute in a IMT performed in a single-stage fashion.Tutkimuksen tausta ja tavoite Luupuutos voi olla seurausta vammasta, infektiosta, kasvaimesta tai näiden kirurgisesta hoidosta. Luupuutosten hoitomenetelmät ovat vakiintumattomat. Luupuutoksia on perinteisesti hoidettu luusiirteillä, verisuonitetuilla luusiirteillä, ulkoisella kiinnityslaitteella tapahtuvalla veto-osteogeneesilla tai keinonivelratkaisuin. Uusista hoitomenetelmistä indusoitu kalvo -tekniikan (Masquelet-tekniikka) on esitetty olevan toimiva pitkien luiden luupuutoksissa. Masquelet-tekniikka on kaksivaiheinen leikkaustekniikka, jonka ensimmäisessä vaiheessa luupuutosalue täytetään PMMA (polymetyylimetakrylaatti) -luusementillä, joka kehittää vierasesinereaktion avulla luupuutosalueen ympärille ns. indusoidun kalvon. Muodostuttuaan kalvo ohjaa verisuonitusta, toimii kantasolujen lähteenä, sekä tuottaa kasvutekijöitä luupuutosalueelle. Leikkaustekniikan toisessa vaiheessa kalvo avataan ja PMMA-luusementti poistetaan, jonka jälkeen kalvon sisään asetetaan luusiirrettä täyttämään luupuutosalue. Olemme kehittäneet BAG-S53P4 -biolasi rakeita käyttäen keinoluusiirteen, jonka avulla mahdollistetaan yksivaiheinen Masquelet-tekniikka ilman erillistä tarvetta luusiirteelle. Lisäksi osa biolaseista on päällystetty poly(DL-lactide-co-glycolide) (PLGA) -polymeerillä, jonka avulla lisätään biolasin mekaanisia kestävyysominaisuuksia, sekä mahdollisesti indusoidaan bioaktiivisempi kalvo verrattuna ei-päällystettyyn biolasiin. Tämän tutkimuksen tavoitteena on osoittaa että BAG-S53P4(±PLGA) biolasi voi kehittää luupuutosalueen ympärille bioaktiivisen kalvon, joka on verrattavissa PMMA-luusementin kehittämään kalvoon. Lisäksi tutkimuksen tavoitteena on todentaa että BAG-S53P4(±PLGA) biolasin sisään muodostuu luukasvua. Aineisto ja menetelmät Tämä tutkimus koostuu soluviljely- ja koe-eläintutkimuksista. Soluviljelytutkimuksessa selvitimme BAG-S53P4(±PLGA) -biolasin vaikutuksia tulehdusreaktioon sekä kantasolujen erilaistumiseen luuta muodostaviksi soluiksi. Koe-eläintutkimuksen tavoitteena oli selvittää indusoituneen kalvon ominaisuuksia sekä luun muodostumista BAG-S53P4(±PLGA) biolasin sisälle luupuutosmallissa. Tulokset BAG-S53P4 -biolasilla todettiin olevan sekä selvä tulehdusreaktiota muokkaava että luunmuodostusta stimuloiva vaikutus. Päällystämällä BAG-S53P4 -biolasi PLGA:lla em. vaikutukset vaimenivat. BAG-S53P4(±PLGA) -biolasin ympärille muodostuneen kalvon todettiin olevan verrattavissa PMMA-indusoituun kalvoon. Lisäksi osoitimme selvää uudisluumuodostusta luupuutosalueella sijaitsevan BAG-S53P4(±PLGA) -biolasin sisällä. Yhteenveto Tutkimuksessa osoitimme, että BAG-S53P4(±PLGA) -biolasi tukee luunmuodostusta luupuutosalueella, kykenee muodostamaan tehokkaan bioaktiivisen kalvon luupuutosalueen ympärille, kykenee stimuloimaan kantasoluja kypsymään luutamuodostaviksi soluiksi, sekä muokkaamaan luupuutoalueen tulehdusvastetta. Täten BAG-S53P4(±PLGA) -biolasilla on tarvittavat ominaisuudet mahdollistamaan sen käytön keinoluusiirteenä yksivaiheisessa Masquelet-tekniikassa. BAG-S53P4(±PLGA) -biolasia käyttämällä vältetään sekä kaksivaiheinen leikkaus että tarve erilliselle luusiirteen käytölle

In vitro and in vivo dissolution of biocompatible S59 glass scaffolds

Abstract Fabrication of porous tissue-engineering scaffolds from bioactive glasses (BAG) is complicated by the tendency of BAG compositions to crystallize in thermal treatments during scaffold manufacture. Here, experimental biocompatible glass S59 (SiO2 59.7 wt%, Na2O 25.5 wt%, CaO 11.0 wt%, P2O5 2.5 wt%, B2O3 1.3 wt%), known to be resistant to crystallization, was used in sintering of glass granules (300–500 µm) into porous scaffolds. The dissolution behavior of the scaffolds was then studied in vivo in rabbit femurs and under continuous flow conditions in vitro (14 days in vitro/56 days in vivo). The scaffolds were osteoconductive in vivo, as bone could grow into the scaffold structure. Still, the scaffolds could not induce sufficiently rapid bone ingrowth to replace the strength lost due to dissolution. The scaffolds lost their structure and strength as the scaffold necks dissolved. In vitro, S59 dissolved congruently throughout the 14-day experiments, resulting in only a slight reaction layer formation. Manufacturing BAG scaffolds from S59 that retain their amorphous structure was thus possible. The relatively rapid and stable dissolution of the scaffold implies that the glass S59 may have the potential to be used in composite implants providing initial strength and stable, predictable release of ions over longer exposure times. Graphical Abstrac

Evaluation of bone growth around bioactive glass S53P4 by scanning acoustic microscopy co-registered with optical interferometry and elemental analysis

Bioactive glass (BAG) is a bone substitute that can be used in orthopaedic surgery. Following implantation, the BAG is expected to be replaced by bone via bone growth and gradual degradation of the BAG. However, the hydroxyapatite mineral forming on BAG resembles bone mineral, not providing sufficient contrast to distinguish the two in X-ray images. In this study, we co-registered coded-excitation scanning acoustic microscopy (CESAM), scanning white light interferometry (SWLI), and scanning electron microscopy with elemental analysis (Energy Dispersive X-ray Spectroscopy) (SEM-EDX) to investigate the bone growth and BAG reactions on a micron scale in a rabbit bone ex vivo. The acoustic impedance map recorded by the CESAM provides high elasticity-associated contrast to study materials and their combinations, while simultaneously producing a topography map of the sample. The acoustic impedance map correlated with the elemental analysis from SEM-EDX. SWLI also produces a topography map, but with higher resolution than CESAM. The two topography maps (CESAM and SWLI) were in good agreement. Furthermore, using information from both maps simultaneously produced by the CESAM (acoustic impedance and topography) allowed determining regions-of-interest related to bone formation around the BAG with greater ease than from either map alone. CESAM is therefore a promising tool for evaluating the degradation of bone substitutes and the bone healing process ex vivo.Peer reviewe