Nanostructured Biomaterials for Tissue Engineered Bone Tissue Reconstruction

Bone tissue engineering strategies are emerging as attractive alternatives to autografts and allografts in bone tissue reconstruction, in particular thanks to their association with nanotechnologies. Nanostructured biomaterials, indeed, mimic the extracellular matrix (ECM) of the natural bone, creating an artificial microenvironment that promotes cell adhesion, proliferation and differentiation. At the same time, the possibility to easily isolate mesenchymal stem cells (MSCs) from different adult tissues together with their multi-lineage differentiation potential makes them an interesting tool in the field of bone tissue engineering. This review gives an overview of the most promising nanostructured biomaterials, used alone or in combination with MSCs, which could in future be employed as bone substitutes. Recent works indicate that composite scaffolds made of ceramics/metals or ceramics/polymers are undoubtedly more effective than the single counterparts in terms of osteoconductivity, osteogenicity and osteoinductivity. A better understanding of the interactions between MSCs and nanostructured biomaterials will surely contribute to the progress of bone tissue engineering

Properties and clinical relevance of osteoinductive biomaterials

This thesis had two main goals: (¿) to investigate parameters influencing osteoinductive potential of biomaterials in order to unravel the mechanism underlying osteoinduction and (¿¿) to investigate performance of osteoinductive biomaterials orthotopically in order to get insight into their clinical relevance

Augmenting Osseointegration Of Implants Using Bone Marrow Stromal Cells

Introduction: The greatest challenge facing the success of orthopaedic implants is improving their fixation to bone to enhance their longevity. Bone marrow stromal cells (BMSC), are a population of plastic-adherent cells derived from the bone marrow. The main hypothesis of this thesis is that viable BMSC can be applied to implants using a fibrin glue-spray system; and increase bone formation adjacent to the implants and improve bone-implant contact. Methods: The experiments were undertaken in a large animal model. Four scenarios were tested 1) The ability of BMSC to improve implant fixation using models of total hip replacement, massive endoprosthetic replacement and bone defect around pins. 2) The effect of varying cell dosages of BMSC in their ability to produce new bone and improve bone implant contact. 3) The effect of differentiating the BMSC along the osteogenic pathway in their ability to produce new bone and improve bone implant contact. 4) The effect of using semi-permeable barriers around BMSC sprayed on implants to prevent cell migration Results: 1) BMSC sprayed on the surface of implants resulted in increased bone formation in the total hip replacement, massive endoprosthetic replacement and bone defect around pin models. 2) Bone formation was higher with osteogenic 10x106 BMSC (112.67 ± 30.75 mm2) compared to osteogenic 2x106 BMSC (76.84 ± 2.25 mm2). No significant difference was noted in bone formation between undifferentiated 1x105 BMSC (30.76 ± 9.43%) and undifferentiated 10x106 BMSC (28.27 ± 14.64%). 3) Osteogenic differentiated 10x106 BMSC (112.67 ± 30.75 mm2) produced more bone than undifferentiated 10x106 BMSC (58.22 ± 17.22 mm2). 4) Using semipermeable barriers resulted in significantly increased bone formation when undifferentiated 1x105 BMSC (61.32 ± 6.94% vs 30.76 ± 9.43%) or undifferentiated 10x106 BMSC (57.46 ± 4.39% vs 28.27 ± 14.64%) was used. This difference was not noted when osteogenic differentiated 10x106 BMSC was used. The experiments confirm that viable BMSC can be successfully isolated from bone marrow aspiration, differentiated along the osteogenic pathway and sprayed on the surface of various orthopaedic implants to improve bone-implant contact. Conclusion: This technique of using BMSC may be an ideal alternative to improve osseointegration of implants in challenging clinical scenarios with deficient bone stock

Enhancing the fixation of massive implants using bone marrow stromal cells.

Previous studies have shown that increased bone growth over massive prosthesis, promoted by hydroxyapatite (HA)-coated collars, can reduce aseptic loosening. Bone tissue engineering techniques using bone marrow stromal cells (BMSCs) may be able to further enhance bone growth and fixation of implants to host bone. The hypothesis of this study was that BMSCs could enhance bone growth and bone-implant contact around bone tumour replacements. Two sources of bone marrow stem cells were firstly investigated, including those isolated directly from ovine bone marrow (BMSCs), and those isolated from ovine peripheral blood (peripheral blood-derived bone marrow stromal-like cells, or PBSCs). PBSCs were isolated after mobilisation via induced blood loss, or treatment with granulocyte-colony stimulating factor (G-CSF). BMSCs and PBSCs were characterised in vitro. A significant increase of fibroblastic colony-forming units (CFU-F) post-G-CSF treatment was observed only after white blood cell counts returned to normal levels, suggesting a possible steady-state balance between haematopoietic stem cells and BMSCs. Ovine BMSCs (oBMSCs), were found to survive and proliferate in fibrin glue or pressurised spray application. An in vivo mid-shaft tibial replacement model was then used to test the effect of autologous oBMSCs in fibrin glue on bone growth and bone-implant contact, when sprayed onto the HA-coated collars, compared to non-treated implants. Radiography showed that the oBMSCs more than doubled the amount of bone growth around the collars of the implants after six months (p=0.017 in the ML view, and p=0.05 in the AP view). Using histological techniques it was shown that bone area was significantly increased (p=0.02). Application of oBMSCs also reduced the radiolucent lines present between the new bone and implants, and improved bone-implant contact. This study demonstrated the potential of BMSCs to augment bone growth and bone-implant contact in conjunction with massive implants. The second in vivo study investigated the effect of BMSC cell dosage and use of allogeneic cells on new bone formation and bone-implant contact in a tibial transcortical pin model in sheep. Partially-HA-coated screws were sprayed with varying concentrations of autologous and allogeneic oBMSCs suspended in fibrin glue, and implanted. After six weeks, no significant difference in bone formation around the pins was found between groups (p>0.05), although the untreated group with HA coating-only had a significant increase in bone formation (p=0.03) compared to the other groups. In conclusion, this project has shown that ovine multipotent BMSCs and PBSCs can be isolated and expanded. When sprayed onto the HA-coated collars of massive implants, BMSCs can augment bone formation and bone-implant contact. However, another model spraying oBMSCs onto trans-cortical pins did not produce a significant increase in bone growth or bone-implant contact. The findings presented may have important clinical applications in the use of BMSCs to reduce aseptic loosening, which may improve the survival of massive implants

An investigation of the osteoinductive properties of Colloss.

Colloss is a sterile extract of bovine bone matrix which consists predominantly of collagen type 1, but also contains several growth factors associated with bone matrix. The ability of Colloss to stimulate bone formation adjacent to titanium implants in vivo and its effect on the proliferation and differentiation of multipotential human bone marrow stromal stem cells (BMSSCs) in vitro and on titanium and hydroxyapatite (HA) was investigated. When BMSSCs were cultured with a solution of Colloss in vitro for 21 days, evidence of osteogenic differentiation was signified by the expression of Alkaline Phosphatase (ALP), collagen type I, calcium deposition and changes in cell morphology. When BMSSCs were cultured on titanium pre-treated with Colloss, there was increased expression of the osteoblastic markers ALP at day 7 and osteopontin on days 14 and 21 compared to BMSSCs cultured on untreated titanium. BMSSCs cultured on porous hydroxyapatite pre-treated with Colloss showed no difference in the expression of the osteogenic markers ALP and osteopontin compared to BMSCCs cultured on untreated porous hydroxyapatite. However there was an increase in collagen type I synthesis, which was 3 times that of control cultures of BMSSCs on untreated HA at 21 days. When Colloss was used to fill 1 and 2 mm defects adjacent to shot blasted titanium implants placed in tibia of sheep, there was no difference in the area of new bone formation or the degree of mineralisation of bone between the control and Colloss containing defects. These findings show that Colloss can induce the osteogenic differentiation of bone marrow stromal stem cells cultured in monolayer and on smooth polished titanium. Results of BMSSCs cultured on hydroxyapatite were inconclusive, although evidence of collagen type I synthesis was suggestive of matrix deposition. Colloss did not enhance bone formation within defects adjacent to titanium implants in vivo

Genome Wide assessment of Early Osseointegration in Implant-Adherent Cells

Objectives: To determine the molecular processes involved in osseointegration. Materials and methods: A structured literature review concerning in vitro and in vivo molecular assessment of osseointegration was performed. A rat and a human model were then used to identify the early molecular processes involved in osseointegration associated with a micro roughened and nanosurface superimposed featured implants. In the rat model, 32 titanium implants with surface topographies exhibiting a micro roughened (AT-II) and nanosurface superimposed featured implants (AT-I) were placed in the tibiae of 8 rats and subsequently harvested at 2 and 4 days after placement. Whereas in the human model, four titanium mini-implants with either a moderately roughened surface (TiOblast) or super-imposed nanoscale topography (Osseospeed) were placed in edentulous sites of eleven systemically healthy subjects and subsequently removed after 3 and 7 days. Total RNA was isolated from cells adherent to retrieved implants. A whole genome microarray using the Affymetrix 1.1 ST Array platform was used to describe the gene expression profiles that were differentially regulated by the implant surfaces. Results: The literature review provided evidence that particular topographic cues can be specifically integrated among the many extracellular signals received by the cell in its signal transduction network. In the rat model, functionally relevant categories related to ossification, skeletal system development, osteoblast differentiation, bone development and biomineral tissue development were upregulated and more prominent at AT-I compared to AT-II. In the human model, there were no significant differences when comparing the two-implant surfaces at each time point. However, the microarray identified several genes that were differentially regulated at day 7 vs. day 3 for both implant surfaces. Functionally relevant categories related to the extracellular matrix, collagen fibril organization and angiogenesis were upregulated at both surfaces. Abundant upregulation of several differential markers of alternative activated macrophages was also observed. The biological processes involved with the inflammatory/immune response gene expression were concomitantly downregulated. Conclusions: The presence of micro-roughened and nanosurface features modulated in vivo bone response. This work confirms previous evaluations and further implicates modulation of the inflammatory/immune responses as a factor affecting the accrual of bone mass shortly after implant placement.Doctor of Philosoph

Granulation tissue formation -The effect of hydroxyapatite coating of cellulose on cellular differentiation

Haavan jyväiskudoksen muodostuminen – Hydroksiapatiittipinnoi-tetun selluloosasienen vaikutus solujen erilaistumiseen paranemisprosessin aikana Etsittäessä uusia luun bioyhteensopivia täytemateriaaleja selluloosasieni päällystettiin luun koostumusta muistuttavalla runsaasti piitä sisältävällä hydroksiapatiittikerroksella. Vastoin odotuksia hydroksiapatiittipinnoitettu selluloosa ei parantanut luun kasvua, vaan päinvastoin ylläpiti tulehdusta ja sidekudossolujen hakeutumista vamma-alueelle. Ihon alle implantoituna sama sienimateriaali edisti merkittävästi haavan verekkään jyväiskudoksen kasvua. Tämän löydöksen perusteella hydroksiapatiittipinnoitetun selluloosasienen vaikutusta haavan soluihin paranemisprosessin aikana tutkittiin tarkemmin ja havaittiin, että tulehdussolujen lisäksi sieniin kertyi tavallista enemmän sekä hematopoieettisia että mesenkymaalisia kantasoluja. Hematopoieettiset kantasolut sijaitsevat luuytimessä lähellä luun sisäpintaa. Luun hydroksiapatiitista vapautuu kalsiumioneja luun jatkuvan fysiologisen uudismuodostuksen ja hajottamisen yhteydessä. Kantasolut etsiytyvät luuytimeen kalsiumia aistivien reseptorien välityksellä. Koska luun pintakerrosta muistuttavasta hydroksiapatiittipinnoitteesta vapautuu kalsiumia, tämän ajateltiin toimivan selityksenä sille, että hematopoieettiset kantasolut hakeutuvat runsaslukuisesti juuri hydroksiapatiittipinnoitettuihin selluloosasieniin. Tämän hypoteesin mukaisesti hydroksiapatiittipinnoitettujen selluloosapalkkien läheisyydestä löydettiin suuria määriä kalsiumreseptoreja sisältäviä soluja. Jatkotutkimuksissa todettiin lisäksi, että hematopoieettiset kantasolut pystyivät sienissä erilaistumaan hemoglobiinia tuottaviksi soluiksi. Havaittujen punasolulinjan merkkiaineiden perusteella näyttäisikin siltä, että haavan paranemiskudoksessa tapahtuu paranemisen aikana ekstramedullaarista erytropoieesia. Nämä soluja ohjaavat vaikutukset saattavat olla hyödyllisiä vaikeasti paranevien haavojen hoidossa.Cellulose was coated with a silica-rich hydroxyapatite layer resembling the mineral composition of bone in search for a possible bone filler material. The hydroxyapatite-coated cellulose did not, however, promote bone repair but instead favored inflammation and fibroplasia. When implanted subcutaneously, these sponges rapidly generated a highly vascular granulation tissue. Further investigation revealed that hydroxyapatite-coated cellulose attracted not only inflammatory cells but also stem cells of both hematopoietic and mesenchymal origin. In the bone marrow, the hematopoietic stem cells reside near the endosteal surface of bone, where the calcium concentration is more than 20-fold of that observed in serum due to bone remodeling by osteoclasts. The hematopoietic stem cells are known to attach to their niche via calcium sensing receptors. The presence and release of calcium ions from the hydroxyapatite layer of the coated sponges might offer an explanation for more abundant accumulation of hematopoietic stem cells to the hydroxyapatite coated implants. Indeed, calcium sensing receptor-positive cells were found especially near the apatite-coated cellulose fibers in the implants. Further analyses indicated that the hematopoietic stem cells were able to differentiate into hemoglobin expressing cells. The presence of erythroid cell markers in the sponges suggests that granulation tissue is capable of extramedullary erythropoiesis. These cell-guiding properties of HA coated cellulose might be utilized in impaired wound healing situations.Siirretty Doriast

Análisis de la capacidad de osteointegración de diferentes revestimientos de superficie de implantes dentales de titanio

La presente tesis doctoral, presentada por compendio de publicaciones, tuvo como objetivo evaluar la dinámica de cicatrización ósea alrededor de implantes dentales con diferentes recubrimientos y tratamientos superficiales, y compararla con implantes con superficie grabada convencional sin tratamiento adicional. Está basada en cuatro artículos publicados en revistas con factor de impacto, del primer cuartil. Todos los artículos giran entorno a los revestimientos y funcionalización de las superficies de los implantes dentales de titanio ya que, en la actualidad, los revestimientos de superficie se han transformado en una de las líneas de investigación con mayor atractivo para los investigadores del sector dental. El primer estudio, evaluó la osteointegración y la formación ósea a diferentes niveles, de los implantes de titanio recubiertos con quitosano, en la mandíbula de un modelo de canino, observándose que, en los implantes recubiertos con quitosano, el análisis de la tendencia en el grupo experimental mostró una significación estadística considerable, en comparación con el grupo control. El segundo, analizó el efecto de la BMP-7 sobre los parámetros biológicos relacionados con la osteointegración de implantes dentales de titanio, con superficies tratadas mediante ácido carboxietilfosfónico. Los resultados preliminares de este estudio preclínico mostraron que, al cabo de cuatro semanas, los implantes experimentales con superficie tratada con ácido carboxietilfosfónico, funcionalizado con BMP-7, obtuvieron diferencias estadísticamente significativas para los parámetros histomorfométricos BIC (Bone Implant Contact) y BIC corregido, en comparación con el grupo de control; sin embargo, no se encontraron diferencias entre los grupos para la formación de hueso nuevo y la densidad ósea. El tercer artículo se centró en una visión general de los conceptos y teorías actuales de la osteointegración y las superficies y recubrimientos actuales de los implantes dentales de titanio, con especial atención a la investigación in vivo, de los implantes recubiertos con quitosano y una perspectiva actual sobre el futuro de los recubrimientos de los implantes dentales de titanio; además se describió un novedoso método de revestimiento, en vías de patentado. El cuarto artículo es una revisión sistemática y metaanálisis, sobre la efectividad de las superficies bioactivas en la osteointegración de los implantes dentales de titanio, con la conclusión que la modificación de las superficies de los implantes, mediante moléculas bioactivas orgánicas, aunque parece favorecer la osteointegración en las primeras fases de cicatrización, no aporta pruebas definitivas de ello