Development of newly conceived biomimetic nano-structured biomaterials as scaffolds for bone and osteochondral regeneration

The present research thesis was focused on the development of new biomaterials and devices for application in regenerative medicine, particularly in the repair/regeneration of bone and osteochondral regions affected by degenerative diseases such as Osteoarthritis and Osteoporosis or serious traumas. More specifically, the work was focused on the synthesis and physico-chemical-morphological characterization of: i) a new superparamagnetic apatite phase; ii) new biomimetic superparamagnetic bone and osteochondral scaffolds; iii) new bioactive bone cements for regenerative vertebroplasty. The new bio-devices were designed to exhibit high biomimicry with hard human tissues and with functionality promoting faster tissue repair and improved texturing. In particular, recent trends in tissue regeneration indicate magnetism as a new tool to stimulate cells towards tissue formation and organization; in this perspective a new superparamagnetic apatite was synthesized by doping apatite lattice with di-and trivalent iron ions during synthesis. This finding was the pin to synthesize newly conceived superparamagnetic bone and osteochondral scaffolds by reproducing in laboratory the biological processes yielding the formation of new bone, i.e. the self-assembly/organization of collagen fibrils and heterogeneous nucleation of nanosized, ionically substituted apatite mimicking the mineral part of bone. The new scaffolds can be magnetically switched on/off and function as workstations guiding fast tissue regeneration by minimally invasive and more efficient approaches. Moreover, in the view of specific treatments for patients affected by osteoporosis or traumas involving vertebrae weakening or fracture, the present work was also dedicated to the development of new self-setting injectable pastes based on strontium-substituted calcium phosphates, able to harden in vivo and transform into strontium-substituted hydroxyapatite. The addition of strontium may provide an anti-osteoporotic effect, aiding to restore the physiologic bone turnover. The ceramic-based paste was also added with bio-polymers, able to be progressively resorbed thus creating additional porosity in the cement body that favour cell colonization and osseointegration.

Design and fabrication of biocompatible scaffolds for the regeneration of tissues

Regenerative medicine and tissue engineering attempt to repair or improve the biological functions of tissues that have been damaged or have ceased to perform their role through three main components: a biocompatible scaffold, cellular component and bioactive molecules. Nanotechnology provide a toolbox of innovative scaffold fabrication procedures in regenerative medicine. In fact, nanotechnology, using manufacturing techniques such as conventional and unconventional lithography, allows fabricating supports with different geometries and sizes as well as displaying physical chemical properties tunable over different length scales. Soft lithography techniques allow to functionalize the support by specific molecules that promote adhesion and control the growth of cells. Understanding cell response to scaffold, and viceversa, is a key issue; here we show our investigation of the essential features required for improving the cell-surface interaction over different scale lengths. The main goal of this thesis has been to devise a nanotechnology-based strategy for the fabrication of scaffolds for tissue regeneration. We made four types of scaffolds, which are able to accurately control cell adhesion and proliferation. For each scaffold, we chose properly designed materials, fabrication and characterization techniques.

Stability of Contour Augmentation and Esthetic Outcomes of Implant Supported Single Crowns in the Esthetic Zone. 3-Year Results of a Prospective Study With Early Implant Placement Post Extraction

Early implant placement is one of the treatment options after tooth extraction. Implant surgery is performed after a healing period of 4 to 8 weeks and combined with a simultaneous contour augmentation using the guided bone regeneration technique to rebuild stable esthetic facial hard- and soft-tissue contours.

Cartilage tissue engineering for degenerative joint disease

Pain in the joint is often due to cartilage degeneration and represents a serious medical problem affecting people of all ages. Although many, mostly surgical techniques, are currently employed to treat cartilage lesions, none has given satisfactory results in the long term. Recent advances in biology and material science have brought tissue engineering to the forefront of new cartilage repair techniques. The combination of autologous cells, specifically designed scaffolds, bioreactors, mechanical stimulations and growth factors together with the knowledge that underlies the principles of cell biology offers promising avenues for cartilage tissue regeneration. The present review explores basic biology mechanisms for cartilage reconstruction and summarizes the advances in the tissue engineering approaches. Furthermore, the limits of the new methods and their potential application in the osteoarthritic conditions are discussed.

Ridge augmentation by applying bioresorbable membranes and deproteinized bovine bone mineral: a report of twelve consecutive cases

OBJECTIVE: Lateral ridge augmentations are traditionally performed using autogenous bone grafts to support membranes for guided bone regeneration (GBR). The bone-harvesting procedure, however, is accompanied by considerable patient morbidity. AIM: The aim of the present study was to test whether or not resorbable membranes and bone substitutes will lead to successful horizontal ridge augmentation allowing implant installation under standard conditions. MATERIAL AND METHODS: Twelve patients in need of implant therapy participated in this study. They revealed bone deficits in the areas intended for implant placement. Soft tissue flaps were carefully raised and blocks or particles of deproteinized bovine bone mineral (DBBM) (Bio-Oss) were placed in the defect area. A collagenous membrane (Bio-Gide) was applied to cover the DBBM and was fixed to the surrounding bone using poly-lactic acid pins. The flaps were sutured to allow for healing by primary intention. RESULTS: All sites in the 12 patients healed uneventfully. No flap dehiscences and no exposures of membranes were observed. Nine to 10 months following augmentation surgery, flaps were raised in order to visualize the outcomes of the augmentation. An integration of the DBBM particles into the newly formed bone was consistently observed. Merely on the surface of the new bone, some pieces of the grafting material were only partly integrated into bone. However, these were not encapsulated by connective tissue but rather anchored into the newly regenerated bone. In all of the cases, but one, the bone volume following regeneration was adequate to place implants in a prosthetically ideal position and according to the standard protocol with complete bone coverage of the surface intended for osseointegration. Before the regenerative procedure, the average crestal bone width was 3.2 mm and to 6.9 mm at the time of implant placement. This difference was statistically significant (P<0.05, Wilcoxon's matched pairs signed-rank test). CONCLUSION: After a healing period of 9-10 months, the combination of DBBM and a collagen membrane is an effective treatment option for horizontal bone augmentation before implant placement.

Early implant placement following single-tooth extraction in the esthetic zone: biologic rationale and surgical procedures

Early implant placement is one treatment option for implant therapy following single-tooth extraction in the anterior maxilla. The surgical technique presented here is characterized by tooth extraction without flap elevation, a 4- to 8-week soft tissue healing period, implant placement in a correct three-dimensional position, simultaneous contour augmentation on the facial aspect with guided bone regeneration using a bioabsorbable collagen membrane combined with autogenous bone chips and a low-substitution bone filler, and tension-free primary wound closure. The surgical step-by-step procedure is presented with a case report. In addition, the biologic rationale is discussed.

Immediate transmucosal implant placement in molar extraction sites: a 12-month prospective multicenter cohort study

AIM: To assess the clinical and radiographic outcomes of immediate transmucosal placement of implants into molar extraction sockets. STUDY DESIGN: Twelve-month multicenter prospective cohort study. MATERIAL AND METHODS: Following molar extraction, tapered implants with an endosseous diameter of 4.8 mm and a shoulder diameter of 6.5 mm were immediately placed into the sockets. Molars with evidence of acute periapical pathology were excluded. After implant placement and achievement of primary stability, flaps were repositioned and sutured allowing a non-submerged, transmucosal healing. Peri-implant marginal defects were treated according to the principles of guided bone regeneration (GBR) by means of deproteinized bovine bone mineral particles in conjunction with a bioresrobable collagen membrane. Standardized radiographs were obtained at baseline and 12 months thereafter. Changes in depth and width of the distance from the implant shoulder (IS) and from the alveolar crest (AC) to the bottom of the defect (BD) were assessed. RESULTS: Eighty-two patients (42 males and 40 females) were enrolled and followed for 12 months. They contributed with 82 tapered implants. Extraction sites displayed sufficient residual bone volume to allow primary stability of all implants. Sixty-four percent of the implants were placed in the areas of 36 and 46. GBR was used in conjunction with the placement of all implants. No post-surgical complications were observed. All implants healed uneventfully yielding a survival rate of 100% and healthy soft tissue conditions after 12 months. Radiographically, statistically significant changes (P<0.0001) in mesial and distal crestal bone levels were observed from baseline to the 12-month follow-up. CONCLUSIONS: The findings of this 12-month prospective cohort study showed that immediate transmucosal implant placement represented a predictable treatment option for the replacement of mandibular and maxillary molars lost due to reasons other than periodontitis including vertical root fractures, endodontic failures and caries.

Prosthetically driven, computer-guided implant planning for the edentulous maxilla: a model study

OBJECTIVES: To analyze computer-assisted diagnostics and virtual implant planning and to evaluate the indication for template-guided flapless surgery and immediate loading in the rehabilitation of the edentulous maxilla. MATERIALS AND METHODS: Forty patients with an edentulous maxilla were selected for this study. The three-dimensional analysis and virtual implant planning was performed with the NobelGuide software program (Nobel Biocare, Göteborg, Sweden). Prior to the computer tomography aesthetics and functional aspects were checked clinically. Either a well-fitting denture or an optimized prosthetic setup was used and then converted to a radiographic template. This allowed for a computer-guided analysis of the jaw together with the prosthesis. Accordingly, the best implant position was determined in relation to the bone structure and prospective tooth position. For all jaws, the hypothetical indication for (1) four implants with a bar overdenture and (2) six implants with a simple fixed prosthesis were planned. The planning of the optimized implant position was then analyzed as follows: the number of implants was calculated that could be placed in sufficient quantity of bone. Additional surgical procedures (guided bone regeneration, sinus floor elevation) that would be necessary due the reduced bone quality and quantity were identified. The indication of template-guided, flapless surgery or an immediate loaded protocol was evaluated. RESULTS: Model (a) - bar overdentures: for 28 patients (70%), all four implants could be placed in sufficient bone (total 112 implants). Thus, a full, flapless procedure could be suggested. For six patients (15%), sufficient bone was not available for any of their planned implants. The remaining six patients had exhibited a combination of sufficient or insufficient bone. Model (b) - simple fixed prosthesis: for 12 patients (30%), all six implants could be placed in sufficient bone (total 72 implants). Thus, a full, flapless procedure could be suggested. For seven patients (17%), sufficient bone was not available for any of their planned implants. The remaining 21 patients had exhibited a combination of sufficient or insufficient bone. DISCUSSION: In the maxilla, advanced atrophy is often observed, and implant placement becomes difficult or impossible. Thus, flapless surgery or an immediate loading protocol can be performed just in a selected number of patients. Nevertheless, the use of a computer program for prosthetically driven implant planning is highly efficient and safe. The three-dimensional view of the maxilla allows the determination of the best implant position, the optimization of the implant axis, and the definition of the best surgical and prosthetic solution for the patient. Thus, a protocol that combines a computer-guided technique with conventional surgical procedures becomes a promising option, which needs to be further evaluated and improved.

Impaired liver regeneration in inducible nitric oxide synthasedeficient mice

The mechanisms that permit adult tissues to regenerate when injured are not well understood. Initiation of liver regeneration requires the injury-related cytokines, tumor necrosis factor (TNF) α and interleukin (IL) 6, and involves the activation of cytokine-regulated transcription factors such as NF-κβ and STAT3. During regeneration, TNFα and IL-6 promote hepatocyte viability, as well as proliferation, because interventions that inhibit either cytokine not only block hepatocyte DNA synthesis, but also increase liver cell death. These observations suggest that the cytokines induce hepatoprotective factors in the regenerating liver. Given evidence that nitric oxide can prevent TNF-mediated activation of the pro-apoptotic protease caspase 3 and protect hepatocytes from cytokine-mediated death, cytokine-inducible nitric oxide synthase (iNOS) may be an important hepatoprotective factor in the regenerating liver. In support of this hypothesis we report that the hepatocyte proliferative response to partial liver resection is severely inhibited in transgenic mice with targeted disruption of the iNOS gene. Instead, partial hepatectomy is followed by increased caspase 3 activity, hepatocyte death, and liver failure, despite preserved induction of TNFα, IL-6, NF-κβ, and STAT3. These results suggest that during successful tissue regeneration, injury-related cytokines induce factors, such as iNOS and its product, NO, that protect surviving cells from cytokine-mediated death.

Polycaprolactone fibres as a potential delivery system for collagen to support bone regeneration

Poly(e-caprolactone) (PCL) is biocompatible, non-immunogenic and non-toxic, and slowly degrades, allowing sufficient time for tissue regeneration. PCL has the potential for application in bone and cartilage repair as it may provide the essential structure required for bone regeneration, however, an ideal scaffold system is still undeveloped. PCL fibres were prepared using the gravity spinning technique, in which collagen was either incorporated into or coated onto the 'as-spun' fibres, in order to develop novel biodegradable polymer fibres which will effectively deliver collagen and support the attachment and proliferation of human osteoblast (HOB) cells for bone regeneration. The physical and mechanical characteristics and cell fibre interactions were analysed. The PCL fibres were found to be highly flexible and inclusion of collagen did not alter the mechanical properties of PCL fibres. Overall, HOB cells were shown to effectively adhere and proliferate on all fibre platforms tested, although proliferation rates were enhanced by surface coating PCL fibres with collagen compared to PCL fibres incorporating collagen and PCL-only fibres. These findings highlight the potential of using gravity spun PCL fibres as a delivery platform for extracellular matrix proteins, such as collagen, in order to enhance cell adherence and proliferation for tissue repair.

Decellularization of bovine corneas for tissue engineering applications

Scaffolds derived from processed tissues offer viable alternatives to synthetic polymers as biological scaffolds for regenerative medicine. Tissue-derived scaffolds provide an extracellular matrix (ECM) as the starting material for wound healing and the functional reconstruction of tissues, offering a potentially valuable approach for the replacement of damaged or missing tissues. Additionally, acellular tissue may provide a natural microenvironment for host-cell migration and the induction of stem cell differentiation to contribute to tissue regeneration. There are a number of processing methods that aim to stabilize and provide an immunologically inert tissue scaffold. Furthermore, these tissue-processing methods can often be applied to xenogenic transplants because the essential components of the ECM are often maintained between species. In this study, we applied several tissue-processing protocols to the cornea in order to obtain a decellularized cornea matrix that maintained the clarity and mechanical properties of the native tissue. Histology, mechanical testing and electron microscopy techniques were used to assess the cell extraction process and the organization of the remaining ECM. In vitro cell seeding experiments confirmed the processed corneas’ biocompatibility.

Sea organisms as a source of collagen-derived polymers for skin tissue engineering applications

Advanced therapies combating acute and chronic skin wounds are likely to be brought about using our knowledge of regenerative medicine coupled with appropriately tissue engineered skin substitutes. At the present time, there are no models of an artificial skin that completely replicate normal uninjured skin and they are usually accompanied by fibrotic reactions that result in the production of a scar. Natural biopolymers such as collagen have been a lot investigated as potential source of biomaterial for skin replacement in Tissue Engineering. Collagens are the most abundant high molecular weight proteins in both invertebrate and vertebrate organisms, including mammals, and possess mainly a structural role in connective tissues. From this, they have been elected as one of the key biological materials in tissue regeneration approaches, as skin tissue engineering. In addition, industry is constantly searching for new natural sources of collagen and upgraded methodologies for their production. The most common sources are skin and bone from bovine and porcine origin. However, these last carry high risk of bovine spongiform encephalopathy or transmissible spongiform encephalopathy and immunogenic responses. On the other hand, the increase of jellyfish has led us to consider this marine organism as potential collagen source for tissue engineering applications. In the present study, novel form of acid and pepsin soluble collagen were extracted from dried Rhopilema hispidum jellyfish species in an effort to obtain an alternative and safer collagen. We studied different methods of collagen purification (tissues and experimental procedures). The best collagen yield was obtained using pepsin extraction method (34.16 mg collagen/g of tissue). The isolated collagen was characterized by SDS-polyacrylamide gel electrophoresis and circular dichroism spectroscopy.

Tissue-specific bioscaffolds for adipose-derived stem/stromal cell expansion and delivery

Decellularized adipose tissue (DAT) is a promising biomaterial for soft tissue regeneration, and it provides a highly conducive microenvironment for human adipose-derived stem/stromal cell (ASC) attachment, proliferation, and adipogenesis. This thesis focused on developing techniques to fabricate 3-D bioscaffolds from enzymatically-digested DAT as platforms for ASC culture and delivery in adipose tissue engineering and large-scale ASC expansion. Initial work investigated chemically crosslinked microcarriers fabricated from pepsin-digested DAT as injectable adipo-inductive substrates for ASCs. DAT microcarriers highly supported ASC adipogenesis compared to gelatin microcarriers in a CELLSPIN system, as confirmed by glycerol-3-phosphate dehydrogenase (GPDH) enzyme activity, lipid accumulation, and endpoint RT-PCR. ASCs cultured on DAT microcarriers in proliferation medium also had elevated PPARγ, C/EBPα, and LPL expression which suggested adipo-inductive properties. In vivo testing of the DAT microcarriers exhibited stable volume retention and enhanced cellular infiltration, tissue remodeling, and angiogenesis. Building from this work, non-chemically crosslinked porous foams and bead foams were fabricated from α-amylase-digested DAT for soft tissue regeneration. Foams were stable and strongly supported ASC adipogenesis based on GPDH activity and endpoint RT-PCR. PPARγ, C/EBPα, and LPL expression in ASCs cultured on the foams in proliferation media indicated adipo-inductive properties. Foams with Young’s moduli similar to human fat also influenced ASC adipogenesis by enhanced GPDH activity. In vivo adipogenesis accompanied by a potent angiogenic response and rapid resorption showed their potential use in wound healing applications. Finally, non-chemically crosslinked porous microcarriers synthesized from α-amylase-digested DAT were investigated for ASC expansion. DAT microcarriers remained stable in culture and supported significantly higher ASC proliferation compared to Cultispher-S microcarriers in a CELLSPIN system. ASC immunophenotype was preserved for all expanded groups, with reduced adhesion marker expression under dynamic conditions. DAT microcarrier expansion upregulated ASC expression of early adipogenic (PPARγ, LPL) and chondrogenic (COMP) markers without inducing a mature phenotype. DAT microcarrier expanded ASCs also showed similar levels of adipogenesis and osteogenesis compared to Cultispher-S despite a significantly higher population fold-change, and had the highest level of chondrogenesis among all groups. This study demonstrates the promising use of DAT microcarriers as a clinically relevant strategy for ASC expansion while maintaining multilineage differentiation capacity.

Poly(3-hydroxyoctanoate), a promising new material for cardiac tissue engineering

Cardiac tissue engineering (CTE) is currently a prime focus of research due to an enormous clinical need. In this work, a novel functional material, Poly(3-hydroxyoctanoate), P(3HO), a medium chain length polyhydroxyalkanoate (PHA), produced using bacterial fermentation, was studied as a new potential material for CTE. Engineered constructs with improved mechanical properties, crucial for supporting the organ during new tissue regeneration, and enhanced surface topography, to allow efficient cell adhesion and proliferation, were fabricated. Our results showed that the mechanical properties of the final patches were close to that of cardiac muscle. Biocompatibility of the P(3HO) neat patches, assessed using Neonatal ventricular rat myocytes (NVRM), showed that the polymer was as good as collagen in terms of cell viability, proliferation and adhesion. Enhanced cell adhesion and proliferation properties were observed when porous and fibrous structures were incorporated to the patches. Also, no deleterious effect was observed on the adults cardiomyocytes’ contraction when cardiomyocytes were seeded on the P(3HO) patches. Hence, P(3HO) based multifunctional cardiac patches are promising constructs for efficient CTE. This work will provide a positive impact on the development of P(3HO) and other PHAs as a novel new family of biodegradable functional materials with huge potential in a range of different biomedical applications, particularly CTE, leading to further interest and exploitation of these materials.