Over the past 5 years, the use of titanium implants as temporary anchorage devices (TADs) has become an important tool in clinical orthodontic practices. The use of TADs have provided orthodontists a way of moving teeth against fixed objects rather than against the surrounding teeth, which tend to counteract desired motion. At present, viable attachment of TADs involves direct insertion through gingival tissue and piercing of the bone. Surface modifications such as sandblasted and acid-etched treatment or bone morphogenetic protein surface treatment, however, can be applied to the TADs to promote enhanced osseointegration, thereby allowing the TADs to serve as stable anchors while avoiding bone puncture. In this study, a comparison was made between sandblasted/acid-etched TADs and sandblasted/acid-etched/recombinant human bone morphogenetic protein-2 (rhBMP-2) treated TADs to determine whether rhBMP-2 promotes enhanced osseointegration. A total of 10 rats (4 controls and 6 treated with rhBMP-2) were used in the study, with 1 TAD placed on the skull of each rat. At the end of 6 weeks, the animals were euthanized by carbon dioxide asphyxiation, and bone blocks, each containing a TAD, were prepared for histological examination and biomechanical characterization. The results of this study showed that TADs treated with rhBMP-2 had greater bone formation at the bone-implant interface and an increase in total implant stability.

Master of Science / Department of Clinical Sciences / David E. Anderson / Drug delivery systems for time release of recombinant human bone morphogenetic protein-2 (rhBMP-2) and antibiotics in orthopedic surgeries continue to be developed. Recently, a biodegradable novel polymeric matrix has been developed for this purpose. We hypothesized that impregnation of the matrix with rhBMP-2 would enhance bone healing. The objectives of the study were to characterize elution of rhBMP-2 and two antimicrobials (tigecycline, tobramycin) from the matrix, and bone response to the matrix in the presence or absence of rhBMP-2 and antimicrobials. In vitro elution of tigecycline, tobramycin, and rhBMP-2 from the matrix was investigated. Drug concentration in media were measured on days 1-6, 8, 10, 13, 15, 17, 21, 25, 28, and 30 using high pressure liquid chromatography/mass spectrometry/mass spectrometry (HPLC/MS/MS; antimicrobials) and ELISA (rhBMP-2). In vivo testing was done using a unicortical defect created into each tibia of twenty adult goats. Animals were randomly assigned to one of 5 groups: 1) control (untreated defect); 2) matrix; 3) matrix+ antimicrobials (tigecycline+tobramycin); 4) matrix+rhBMP-2; and 5) matrix+antimicrobials+rhBMP-2. Plasma concentration of tigecycline and tobramycin and serum concentration of rhBMP-2 were measured by the above techniques on days 1-7, 9, 11, 13, 15, 17, 22, 26, and 30. Bone response was assessed on days 0, 14, and 30 using radiographic scoring and dual energy X-ray absorptiometry (bone mineral density [BMD]). After euthanasia on day 30, histomorphologic analyses of the bone defects were done. Categorical variables were analyzed using a generalized linear model, and continuous variables using an ANOVA with P < 0.05 considered significant. In vitro elution was characterized by a rapid release on day 1 followed by a slow release until day 30 for both antimicrobials and rhBMP-2. Plasma antimicrobial concentrations showed continued release throughout the study period. Serum rhBMP-2 concentration, radiographic scores and BMD were not significantly different between groups. Periosteal and endosteal reaction surface areas were significantly greater surrounding the defects in group 4 (matrix+rhBMP-2). There was no significant difference between the groups for the percent of bone filling the defect. The matrix served as an appropriate antimicrobial and rhBMP-2 delivery system and successfully stimulated bone production when rhBMP-2 was present.

Adipositas und Typ-2-Diabetes sind häufige Erkrankungen des Stoffwechsels. Zur Basistherapie der Adipositas und des Typ-2-Diabetes gehören eine gesunde Ernährungs- weise und die Erhöhung der körperlichen Aktivität unter anderem mit dem Ziel der Gewichtsreduktion. Vermehrte Bewegung führt neben der Verbesserung der körperlichen Leistungsfähigkeit zur Fettmassenreduktion, Verbesserungen der Hyperglykämie, Lipo- proteinstoffwechsels und des Adipokinprofils. Bone morphogenetic proteins (BMPs) werden im Fettgewebe produziert und spielen eine wichtige Rolle in der Adipogenese und Transdifferenzierung von Adipozyten. Während ein Zusammenhang zwischen der BMP-7-Serumkonzentration und Adipositas vor kurzem belegt wurde, ist bisher nicht bekannt, ob weitere BMPs wie BMP-2 und -4 mit Adipositas und Typ-2-Diabetes assoziiert sind. Ziel dieser Arbeit war es deshalb zu untersuchen, ob die BMP-2 und -4 Serumkonzentrationen im Zusammenhang mit Körpergewicht, Fett- verteilung und Parametern des Glukosestoffwechsels bei Patienten mit Adipositas und Typ-2-Diabetes (n=213) stehen. Im Rahmen von drei Interventionsstudien wurde der Einfluss einer hypokalorischen Ernährungsweise über sechs Monate (n=19), eines 45,3 ± 7,4 kg Gewichtsverlustes ein Jahr nach bariatrischer Chirurgie (n=32) sowie eines zwölf- wöchigen Trainingsprogramms (n=60) auf die BMP-2- und -4-Serumkonzentrationen untersucht. Zusätzlich wurde die BMP-2-und -4-mRNA-Expression in humanen omentalen und subkutanen Fettgewebsproben von 161 Patienten charakterisiert. Die BMP-2- und -4-Serumkonzentrationen und die BMP-2- und -4-mRNA-Expression im viszeralen Fettgewebe korrelieren signifikant mit dem BMI und dem Körperfettgehalt. Zirkulierende BMP-4-Spiegel sind geschlechtsabhängig und bei Patienten mit T2D signifikant niedriger als bei gesunden Kontrollpatienten. Sowohl eine moderate Gewichts- reduktion durch kalorienreduzierte Ernährung als auch ein Gewichtsverlust von 45,3 ± 7,4 kg nach bariatrischer Chirurgie führen zu einer signifikanten Reduktion der zirkulierenden BMP-2- und -4-Spiegel. Das zwölfwöchige Trainingsprogramm führte lediglich zu einer signifikanten Reduktion der BMP-2-Serumkonzentration und zu signifikanten Ver- besserung der Leistungsfähigkeit, von Parametern des Glukosestoffwechsels und der Serumkonzentrationen von Adiponektin und Interleukin-6. Zusammengefasst zeigen die Daten, dass erhöhte Serumkonzentrationen von BMP-2 und 4 mit Adipositas assoziiert sind und durch Gewichtsreduktion und Erhöhung der körperlichen Aktivität verringert werden können. Die BMP-2- und -4-mRNA-Expression im viszeralen Fettgewebe kann zu erhöhten Serumkonzentrationen dieser Adipokine bei viszeraler Fettverteilung beitragen.

A utilização de osteoindutores como a proteína morfogenética óssea recombinante humana tipo 2 (rhBMP-2) em cirurgia oral e maxilofacial para reparação e regeneração óssea tem aumentado progressivamente. Porém, suas indicações ainda estão limitadas a preenchimento de cavidades e pequenas reconstruções. Este estudo teve como objetivo analisar e comparar, por meio da microtomografia computadorizada tridimensional (Micro-TC 3D) e histomorfometria, a arquitetura óssea, a taxa de osso neoformado e a taxa de biodegradação do -Tricálcio Fostato (-TCP), Fosfato de Cálcio Bifásico (BCP) e Osso Mineral Bovino (BBM), utilizados como carreadores adicionais à rhBMP-2/esponja de colágeno (ACS) em um modelo de regeneração óssea guiada (ROG) vertical em calvária de coelhos. Quatro cilindros de titânio foram fixados à calvária de 20 coelhos da raça Nova Zelândia. No Grupo 1 (n = 10), 3 cilindros foram aleatoriamente preenchidos com um dos materiais teste utilizados como carreadores e um cilindro foi preenchido com coágulo sanguíneo (CO). No Grupo 2 (n = 10), os cilindros foram aleatoriamente designados para os mesmos materiais e coágulo sanguíneo, com a adição da rhBMP-2/ACS. Após 14 semanas de reparação, as amostras foram coletadas e enviadas para a aquisição de imagens da Micro-TC e processamento histológico. De acordo com a análise histomorfométrica, a área óssea média para o Grupo 2 (com rhBMP-2) foi maior do que no Grupo 1 (sem rhBMP-2) para os materiais BCP e -TCP (p <0,001). Não houve diferença entre os grupos para BBM e CO (p> 0,05). Em relação às taxas de reabsorção, a área média dos materiais remanescentes no Grupo 2 foi menor do que no Grupo 1 para todos os materiais (p <0,001) e BBM e -TCP obtiveram a maior taxa de reabsorção nos dois grupos (BBM = -TCP > BCP). A análise da micro-TC revelou que no Grupo 2, BCP e -TCP apresentaram maior volume ósseo médio (BV) do que no Grupo 1 (p <0,05). Não houve diferença entre os grupos para os materiais BBM e CO (p> 0,05). O volume médio de materiais restantes (MV) para o Grupo 2 foi menor do que no Grupo 1 para BBM e -TCP (p <0,05), sem diferença significante entre os grupos para BCP (p = 0,848). A análise dos parâmetros Fator Padrão Trabecular (Tb.Pf) e Índice de Modelo Estrutural (SMI) mostrou no Grupo 2 valores negativos para BCP e -TCP, indicando melhor interconectividade e presença de arquitetura trabecular mais achatada e côncava, indicadores de melhor qualidade e resistência óssea. Pelos resultados apresentados concluiu-se que a utilização da rhBMP-2/ACS associada aos materiais carreadores BCP (Fosfato de Cálcio Bifásico) e -TCP (-Fosfato Tricálcio) aumentou significativamente a formação óssea neste modelo de ROG em calvária de coelhos além de acelerar a biodegradação dos materiais BBM (Osso Mineral Bovino) e -TCP (-Fosfato Tricálcio) neste modelo de ROG. / The use of osteoinductors as the human recombinant bone morphogenetic protein 2 type 2 (rhBMP-2) in oral and maxillofacial surgery for bone regeneration and repair has progressively increased. However, indications are still limited to filling of cavities and small reconstructions. However, its indications are still limited to filling of cavities and small reconstructions. This study aimed to analyze and compare, by means of three-dimensional microtomography (Micro-CT 3D) and histomorphometry, the bone architecture, the rate of newly formed bone and the biodegradation rate of beta tricalcium phosphate (-TCP), biphasic calcium phosphate (BCP), and mineral bovine bone mineral (BBM) used as additional carriers to rhBMP-2/absorbable collagen sponge (ACS) in a vertical guided bone regeneration model (GBR) in rabbit calvarium. Four titanium cylinders were fixed to the calvarium of 22 New Zealand rabbits. In Group 1 (n = 10), 3 cylinders were randomly filled with one of the test materials and 1 cylinder was filled with a blood clot (CL). In Group 2 (n = 10), the cylinders were randomly assigned to the same materials and blood clot, with the addition of rhBMP-2. After 14 weeks of healing the samples were collected and sent to 3D Micro-CT image acquisition and histological processing. According to histomorphometric analysis, the mean bone area in Group 2 (with rhBMP-2) was greater than in Group 1 (without rhBMP-2) to materials BCP and -TCP (p<0.001). There was no difference between groups to BBM and CL (p>0.05). Regarding the resorption rates, the mean area of remaining materials in Group 2 was lower than in Group 1 to all materials (p<0.001) and BBM and -TCP had the greater resorption rate in both groups (BBM= -TCP >BCP). The Micro-CT analysis revealed that in Group 2, BCP and -TCP had greater mean bone volume (BV) than in Group 1 (p<0.05). There was no difference between groups to materials BBM and CL (p>0.05). The mean volume of remaining materials (MV) in Group 2 was lower than in Group 1 to BBM and -TCP (p<0.05). There was no difference between groups to BC (p=0.848). The analysis of the parameters Trabecular Pattern Factor (Tb.Pf) and Structure Model Índex (SMI), in Group 2 showed negative values for BCP and -TCP, indicating better interconnectivity and presence of more plate-like and trabecular architecture more flattened and concave, indicators of better quality and bone strength. By presented results it was concluded that the use of rhBMP-2/ACS associated with the carriers materials biphasic calcium phosphate (BCP) and beta tricalcium phosphate (-TCP) used as additional carriers, significantly increased bone formation in addition to accelerate the resorption of the materials BO (bovine bone mineral) and -TCP in this guided bone regeneration model in rabbit calvaria.

A utilização de osteoindutores como a proteína morfogenética óssea recombinante humana tipo 2 (rhBMP-2) em cirurgia oral e maxilofacial para reparação e regeneração óssea tem aumentado progressivamente. Porém, suas indicações ainda estão limitadas a preenchimento de cavidades e pequenas reconstruções. Este estudo teve como objetivo analisar e comparar, por meio da microtomografia computadorizada tridimensional (Micro-TC 3D) e histomorfometria, a arquitetura óssea, a taxa de osso neoformado e a taxa de biodegradação do -Tricálcio Fostato (-TCP), Fosfato de Cálcio Bifásico (BCP) e Osso Mineral Bovino (BBM), utilizados como carreadores adicionais à rhBMP-2/esponja de colágeno (ACS) em um modelo de regeneração óssea guiada (ROG) vertical em calvária de coelhos. Quatro cilindros de titânio foram fixados à calvária de 20 coelhos da raça Nova Zelândia. No Grupo 1 (n = 10), 3 cilindros foram aleatoriamente preenchidos com um dos materiais teste utilizados como carreadores e um cilindro foi preenchido com coágulo sanguíneo (CO). No Grupo 2 (n = 10), os cilindros foram aleatoriamente designados para os mesmos materiais e coágulo sanguíneo, com a adição da rhBMP-2/ACS. Após 14 semanas de reparação, as amostras foram coletadas e enviadas para a aquisição de imagens da Micro-TC e processamento histológico. De acordo com a análise histomorfométrica, a área óssea média para o Grupo 2 (com rhBMP-2) foi maior do que no Grupo 1 (sem rhBMP-2) para os materiais BCP e -TCP (p <0,001). Não houve diferença entre os grupos para BBM e CO (p> 0,05). Em relação às taxas de reabsorção, a área média dos materiais remanescentes no Grupo 2 foi menor do que no Grupo 1 para todos os materiais (p <0,001) e BBM e -TCP obtiveram a maior taxa de reabsorção nos dois grupos (BBM = -TCP > BCP). A análise da micro-TC revelou que no Grupo 2, BCP e -TCP apresentaram maior volume ósseo médio (BV) do que no Grupo 1 (p <0,05). Não houve diferença entre os grupos para os materiais BBM e CO (p> 0,05). O volume médio de materiais restantes (MV) para o Grupo 2 foi menor do que no Grupo 1 para BBM e -TCP (p <0,05), sem diferença significante entre os grupos para BCP (p = 0,848). A análise dos parâmetros Fator Padrão Trabecular (Tb.Pf) e Índice de Modelo Estrutural (SMI) mostrou no Grupo 2 valores negativos para BCP e -TCP, indicando melhor interconectividade e presença de arquitetura trabecular mais achatada e côncava, indicadores de melhor qualidade e resistência óssea. Pelos resultados apresentados concluiu-se que a utilização da rhBMP-2/ACS associada aos materiais carreadores BCP (Fosfato de Cálcio Bifásico) e -TCP (-Fosfato Tricálcio) aumentou significativamente a formação óssea neste modelo de ROG em calvária de coelhos além de acelerar a biodegradação dos materiais BBM (Osso Mineral Bovino) e -TCP (-Fosfato Tricálcio) neste modelo de ROG. / The use of osteoinductors as the human recombinant bone morphogenetic protein 2 type 2 (rhBMP-2) in oral and maxillofacial surgery for bone regeneration and repair has progressively increased. However, indications are still limited to filling of cavities and small reconstructions. However, its indications are still limited to filling of cavities and small reconstructions. This study aimed to analyze and compare, by means of three-dimensional microtomography (Micro-CT 3D) and histomorphometry, the bone architecture, the rate of newly formed bone and the biodegradation rate of beta tricalcium phosphate (-TCP), biphasic calcium phosphate (BCP), and mineral bovine bone mineral (BBM) used as additional carriers to rhBMP-2/absorbable collagen sponge (ACS) in a vertical guided bone regeneration model (GBR) in rabbit calvarium. Four titanium cylinders were fixed to the calvarium of 22 New Zealand rabbits. In Group 1 (n = 10), 3 cylinders were randomly filled with one of the test materials and 1 cylinder was filled with a blood clot (CL). In Group 2 (n = 10), the cylinders were randomly assigned to the same materials and blood clot, with the addition of rhBMP-2. After 14 weeks of healing the samples were collected and sent to 3D Micro-CT image acquisition and histological processing. According to histomorphometric analysis, the mean bone area in Group 2 (with rhBMP-2) was greater than in Group 1 (without rhBMP-2) to materials BCP and -TCP (p<0.001). There was no difference between groups to BBM and CL (p>0.05). Regarding the resorption rates, the mean area of remaining materials in Group 2 was lower than in Group 1 to all materials (p<0.001) and BBM and -TCP had the greater resorption rate in both groups (BBM= -TCP >BCP). The Micro-CT analysis revealed that in Group 2, BCP and -TCP had greater mean bone volume (BV) than in Group 1 (p<0.05). There was no difference between groups to materials BBM and CL (p>0.05). The mean volume of remaining materials (MV) in Group 2 was lower than in Group 1 to BBM and -TCP (p<0.05). There was no difference between groups to BC (p=0.848). The analysis of the parameters Trabecular Pattern Factor (Tb.Pf) and Structure Model Índex (SMI), in Group 2 showed negative values for BCP and -TCP, indicating better interconnectivity and presence of more plate-like and trabecular architecture more flattened and concave, indicators of better quality and bone strength. By presented results it was concluded that the use of rhBMP-2/ACS associated with the carriers materials biphasic calcium phosphate (BCP) and beta tricalcium phosphate (-TCP) used as additional carriers, significantly increased bone formation in addition to accelerate the resorption of the materials BO (bovine bone mineral) and -TCP in this guided bone regeneration model in rabbit calvaria.

Introduction: Idiopathic Pulmonary arteriole hypertension (IPAH) is a rare but severely debilitating disease that strikes women to men at a ratio of 3:1. Endothelial cell (EC) dysfunction is a hallmark of the disease. This includes rapid growth of the ECs until the occlusion of the vasculature as well as decreased blood levels of vasodilators. Markedly increased levels of endothelin-1, a potent vasoconstrictor and smooth muscle mitogen, have been noted in IPAH patients. / Recently mutations in the bone morphogenic protein receptor type II (BMPRII) have been linked to the disease. Interestingly mutations in activin-like kinase-1 (ALK-1) and endoglin have been linked to hereditary haemorrhagic telangiectasia (HHT), a disease that results in PAH clinically indistinguishable from IPAH. All of these proteins are either receptors or co-receptors to members of the TGFbeta superfamily. The connection of these mutations to the disease still remains largely a mystery to researchers and the effects of either bone morphogenic proteins 2, 4, 7 or TGFbeta levels on endothelin-1(ET-1) production in human microvascular endothelial cells cultured from normal lungs (HMVEC-LBI) are unknown. / Methods: HMVEC-LBI cells were cultured in the presence of various concentrations of BMP 2,4,7 and TGFbeta, in complete media or serum starved conditions. After allotted time points the media was collected and assayed by ELISA, meanwhile the cells were lysed and protein content assayed for normalization purposes. Small Mothers against Decapentaplegic (SMAD) 1/5 phosphorylation was also measured. / Results and Conclusions: Despite evidence that all BMPs used were biologically active, namely through SMAD phosphorylation studies, only BMP7 at very high dosages increased ET-1 production levels. TGFbeta had a more pronounced effect at earlier time points with lower concentrations. The results provide insights on the effects of an important group of proteins, the BMPs and TGFbeta, on lung microvascular ECs and which are likely the key cellular player In IPAH development. These findings may have clinical relevance in terms of control of the disease and understanding the normal response of these cells BMPs and TGFbeta.

Over 600,000 bone-grafting operations are performed each year in the United States. The majority of the bone used for these surgeries comes from autografts that are limited in quantity or allografts with high failure rates. Current synthetic bone grafting materials have poor mechanical properties, handling characteristics, and bioactivity. The goal of this dissertation was to develop a clinically translatable bone tissue engineering scaffold with improved handling characteristics, bioactivity, and smart delivery modalities. We hypothesized that this could be achieved through the rational selection of Food and Drug Administration (FDA) approved materials that blend favorably with hydroxyapatite (HA), the principle mineral component in bone. This dissertation describes the development of smart bone tissue engineering scaffolds composed of the biodegradable amphiphilic polymer poly(D,L-lactic acid-co-ethylene glycol-co- D,L-lactic acid) (PELA) and HA. Electrospun nanofibrous HA-PELA scaffolds exhibited improved handling characteristics and bioactivity over conventional HApoly( D,L-lactic acid) composites. Electrospun HA-PELA was hydrophilic, elastic, stiffened upon hydration, and supported the attachment and osteogenic differentiation of rat bone marrow stromal cells (MSCs). These in vitro properties translated into robust bone formation in vivo using a critical-size femoral defect model in rats. Spiral-wrapped HA-PELA scaffolds, loaded with MSCs or a lowdose of recombinant human bone morphogenetic protein-2, templated bone formation along the defect. As an alternate approach, PELA and HA-PELA were viii rapid prototyped into three-dimensional (3-D) macroporous scaffolds using a consumer-grade 3-D printer. These 3-D scaffolds have differential cell adhesion characteristics, swell and stiffen upon hydration, and exhibit hydration-induced self-fixation in a simulated confined defect. HA-PELA also exhibits thermal shape memory behavior, enabling the minimally invasive delivery and rapid (>3 sec) shape recovery of 3-D scaffolds at physiologically safe temperatures (~ 50ºC). Overall, this dissertation demonstrates how the rational selection of FDA approved materials with synergistic interactions results in smart biomaterials with high potential for clinical translation.

No description available.

Bone is a remarkable multifunctional tissue with the ability to regenerate and remodel without generating any scar tissue. However, bone loss due to injury or diseases can be a great challenge and affect the patient significantly. Autologous bone grafting is commonly used throughout the world. Autograft both fills the void and is bone inductive, housing the particular cells that are needed for bone regeneration. However, a regenerative complement to autograft is of great interest as the use of biomaterials loaded with bioactive molecules can avoid donor site morbidity and the problem of a limited volume of material. Two such regenerative products that utilise bone morphogenetic protein (BMP)-7 and -2 have been used for more than a decade clinically. Unfortunately, several side effects have been reported, such as severe swelling due to inflammation and ectopic bone formation. Additionally, the products require open surgery and use of supra physiological doses of the BMPs due to poor localisation and retention of the growth factor. The purpose of this thesis was to harness the strong inductive capacity of the BMP-2 by optimising the carrier of this bioactive protein, thereby minimising the side effects that are associated with the clinical products and facilitating safe and localised bone regeneration. We focused on an injectable hyaluronan-based carrier developed through polymer chemistry at the University of Uppsala. The strategy was to use the body’s own regenerative pathway to stimulate and enhance bone healing in a manner similar to the natural bone-healing process. The hyaluronan-based carrier has a similar composition to the natural extracellular matrix and is degraded by resident enzymes. Earlier studies have shown improved properties when adding hydroxyapatite, a calcium phosphate that constitutes the inorganic part of the bone matrix. In Paper I, the aim was to improve the carrier by adding other forms of calcium phosphate. The results indicated that bone formation was enhanced when using nano-sized hydroxyapatite. In Paper II, we discovered the importance of crushing the material, thus enhancing permeability and enlarging the surface area. We wished to further develop the carrier system, but were lacking an animal model with relatively high throughput, facilitated access, paired data, and we were also committed to the 3Rs of refinement, reduction, and replacement. To meet these challenges, we developed and refined an animal model, and this is described in Paper III. In Paper IV, we sought to further optimise the biomaterial properties of the hydrogel through covalent bonding of bisphosphonates to the hyaluronan hydrogel. This resulted in exceptional retention of the growth factor BMP-2. In Paper V, SPECT/PET/µCT was combined as a tri-modal imaging method to allow visualisation of the biomaterial’s in situ action, in terms of drug retention, osteoblast activity and mineralisation. Finally, in Paper VI the correlation between existing in vitro results with in vivo outcomes was observed for an array of biomaterials. The study identified a surprisingly poor correlation between in vitro and in vivo assessment of biomaterials for osteogenesis.

Bone is a remarkable multifunctional tissue with the ability to regenerate and remodel without generating any scar tissue. However, bone loss due to injury or diseases can be a great challenge and affect the patient significantly. Transplanting bone graft from one site in the patient to the site of fracture or bone void, i.e. autologous bone grafting is commonly used throughout the world. The transplanted bone not only fills voids, but is also bone inductive, housing the particular cells that are needed for bone regeneration. Nevertheless, a regenerative complement to autograft is of great interest and importance because the benefits from an off-the-shelf product with as good of healing capacity as autograft will circumvent most of the drawbacks with autograft. With a regenerative-medicine approach, the use of biomaterials loaded with bioactive molecules can avoid donor site morbidity and the problem of limited volume of material. Two such regenerative products that utilize bone morphogenetic protein 7 and 2 have been used for more than a decade in the clinic. However, some severe side effects have been reported, such as severe swelling due to inflammation and ectopic bone formation. Additionally, the products require open surgery, use of supra physiological doses of the BMPs due to poor localization and retention of the growth factors. The purpose of this thesis was to harness the strong inductive capability of the BMP-2 by optimizing the carrier of this bioactive protein, thereby minimizing the side effects that are associated with the clinical products and facilitating safe and localized bone regeneration at the desired site. We focused on an injectable hyaluronan-based carrier. The strategy was to use the body’s own regenerative pathway to stimulate and enhance bone healing in a manner similar to the natural bone-healing process. The hyaluronan-based carrier has a similar composition to the natural extracellular matrix and is degraded by resident hyaluronidase enzymes. Earlier studies have shown a more controlled release and improved mechanical properties when adding a weight of 25 percent of hydroxyapatite, a calcium phosphate that constitutes the inorganic part of the bone matrix. In Paper I, the aim was to improve the carrier by adding other forms of calcium phosphate. The results indicated that the bone formation was enhanced when using nano-sized hydroxyapatite. We wished to further develop the carrier system but were lacking an animal model with high output and easy access. We also wanted to provide paired data and were committed to the 3 Rs of refinement, reduction and replacement. To meet these challenges, we developed and refined an animal model, and this is described in Paper II. In Paper III, we characterized and optimized the handling properties of the carrier. In Paper IV, we discovered the importance of crushing the material, thus enhancing permeability and enlarging the surface area. In Paper V, we sought to further optimize biomaterial properties of the hydrogel through covalently bonding of bisphosphonates to the hyaluronan hydrogel. The results demonstrated exceptional retention of the growth factor BMP-2. In Paper VI, the in vivo response related to the release of the growth factor was examined by combining a SPECT/PET/µCT imaging method to visualize both the retention of the drug, and the in-vivo response in terms of mineralization.