• bisphosphonates
• calcium phosphate ceramics as bone substitute
• in vitro study
• in vivo study
• osteoporosis
• strontium

Bone is a dynamic, vascularised tissue with a unique capacity to heal and remodel without leaving a scar. These properties, together with its capacity to rapidly mobilize mineral stores on metabolic demand, make it the ultimate smart material. Its main role is to provide structural support for the body. Furthermore the skeleton also serves as a mineral reservoir, supports muscular contraction resulting in motion, withstands load bearing and protects internal organs. Hence, it is logical to say that major alterations in its structure due to injury or disease can dramatically alter one’s body equilibrium and quality of life. In fact, when the dimension of a lesion overcomes the intrinsic regenerative capacity of bone or when patient related conditions, such as infections, inadequate blood supply or systemic diseases impair bone healing, tissue transplants or biomaterials are necessary to support, stimulate and accelerate the bone ingrowth.
Annually, more than 2.2 million bone grafting procedures are performed worldwide. The current gold standard for bone repair is the use of autologous bone grafts harvested from a remote site in the patient. A few of the many problems associated with autografts include donor site morbidity and the restricted availability. In addition to autografts, success has been reported with the use of allografts. Like the autografts, allografts are limited in supply and risks of disease transmission and immune rejection exist. Despite the benefits of both autografts and allografts, the relative concerns over their use have led to the development of numerous synthetic bone substitutes. Moreover, biomaterials play central roles in modern strategies in regenerative medicine and tissue engineering as designable biophysical and biochemical milieus that direct cellular behavior and function. The guidance provided by biomaterials may facilitate restoration of structure and function of damaged or dysfunctional tissues, both in cell-based therapies, such as those where biomaterials as scaffolds or carriers deliver transplanted cells or matrices induce morphogenesis in bioengineered tissues constructed ex vivo, and in a-cellular therapies, such as those where materials induce recruitment, ingrowth and differentiation of cells from healthy residual tissues in situ. Such materials should provide a provisional three-dimensional (3-D) support to interact bio-molecularly with cells to control their function, guiding the spatially and temporally complex multicellular processes of tissue formation and regeneration.
Both biologically derived and synthetic materials have been extensively explored in regenerative medicine and tissue engineering. In general, materials from biological sources (e.g., purified protein components such as collagens from animal tissues) are advantageous because of their inherent properties of biological recognition, including presentation of receptor-binding ligands and susceptibility to cell-triggered proteolytic degradation and remodelling. Despite these advantages, many issues have spurred the development of synthetic biomaterials as cellular substrates, including complexities associated with purification, immunogenicity and pathogen transmission. Although some of these limitations can be overcome by recombinant protein expression technologies, greater control over materials properties and tissue responses could be achieved where synthetic analogs are available.
One of the most promising group of synthetic bone substitutes are calcium phosphate ceramics (CaPs). The most commonly used CaPs are hydroxyapatite (HA) and tricalcium phosphate (TCP) or an intrinsic combination of the two.
Biocompatibility, bioactivity, and easiness to adapt to the shape of bone cavities and defects are some of the reasons of the increasing interest towards CaPs application in both medical and dental surgery. Preclinical and clinical studies demonstrate the effectiveness of HA and TCP for the treatment of bone defects because of trauma or surgery. TCP is also proposed as bone cement associated to poly-methylmethacrylate (PMMA), the most widely employed material for implant fixation, for the improvement of PMMA biocompatibility. A further advantage of CaPs with respect to PMMA is the absence of heat development during hardening. Mixing of a CaPs powder with an aqueous solution leads to the hardening of the cement paste through a low temperature dissolution/precipitation reaction. The lack of an exothermic reaction is desirable not only to avoid inflammatory tissue response, but also for potential employment of the cements for the delivery of sensitive molecules such as drugs or proteins.
Many strategies exist in order to improve bone lesion treatment and drugs or ion scan be used in association with ceramic materials to accelerate bone repair and remodelling. Bisphosphonates (BPs) are a chemical class of compounds in widespread use since the 1970s for the management of disorders of bone metabolism, associated to bone loss. These compounds bind strongly to HA crystals, they suppress osteoclast-mediated bone resorption, they are retained for a long time in the skeleton, and they are excreted un-metabolized in urine. Also strontium (Sr), that is present in the mineral phase of bone, has the ability to enhance bone volume and prevent bone loss. Furthermore, Sr administration as Sr ranelate has recently been shown to reduce the incidence of fractures in osteoporotic patients. Sr exerts both anti-resorbing and bone forming effects in vitro, because it increases the number of osteoblasts and decreases the number and the activity of osteoclasts.
The aims of this research, in collaboration with the Department of Chemistry, “G. Ciamician”, Bologna University were:
1) to manufacture a biomimetic gelatin- calcium phosphate bone cement and to carry out the preclinical biological studies on healthy bone defects;
2) to develop an Alendronate (AL)- HA nanocomposite material prepared using different concentrations of AL and a Sr- HA nanocrystal prepared from solutions containing different concentrations of Sr atom and to carry out preclinical biological studies on healthy and osteoporotic bone.
Preclinical biological studies were aimed to evaluate:
-in vitro and in vivo biocompatibility (following Organization of Standardization (ISO) guides 10993);
-effects on bone cell behavior;
-preclinical effectiveness in bone defects and in healing of bone in osteoporosis conditions.
Following a biomimetic strategy aimed to reproducing bone characteristics, the first aim of this thesis was to investigate the biological properties of new gelatin enriched calcium phosphate cement (GEL-CP) that exhibits improved mechanical properties with respect to the cement prepared without gelatin (C-CP) and to HA. For this purpose we cultured human osteoblast MG63 on GEL-CP as compared with cells cultured on C-CP and HA sample. Cell attachment, proliferation and differentiation were evaluated up to 21 days. SEM analyses revealed that osteoblasts grown on GEL-CP showed a normal morphology and biological tests demonstrated very good rate of proliferation and viability at each experimental time. The presence of gelatin stimulated alkaline phosphatase activity, collagen I and transforming growth factor ß1 production. This in vitro data indicate that the new cement GEL-CP favors osteoblast proliferation, activation of their metabolism and differentiation. These data have led to believe that GEL-CP could be a promising biomimetic material to be successfully applied as bone substitute. To this purpose, an in vivo investigation was performed on healthy rabbits, in order to perform histological, histomorphometrical and microtomographical analyses on bone healing rate and osseointegration of these materials. Our results showed that at both 4-weeks and 12-weeks after surgery HA implant had significantly higher value of healing rate than GEL-CP implant. No differences were found in osseointegration index between C-CP, GEL-CP and HA groups. These in vivo results have allowed to detect that an improvement in the structure of this novel material would lead to a greater upgrading in the in vivo experimental results.
The second aim of this thesis was to evaluate an HA nanocrystals synthesized at different extent of Sr substitution for calcium and an HA-AL nanocrystals synthesized at increasing AL content. HA nanocrystals were synthesized at Sr contents of 0, 1, 3, 7 atom %. Sr incorporation for calcium (Ca) was confirmed by the linear increase of the unit cell parameters of HA, in agreement with the different ionic radii of the two ions. Moreover, Sr substitution slightly affected HA structural order and the shape of the nanocrystals. Osteoblast-like MG63 cells cultured on the nanocrystals showed good proliferation and increased values of the differentiation parameters. In particular, when cultured on samples with Sr concentration in the range 3–7 atom %, osteoblasts demonstrated an increased values of alkaline phosphatase activity, collagen type I, and osteocalcin production. Moreover, osteoclast number on all the Sr-doped samples was significantly smaller than that on HA, and it decrease on increasing Sr content.
Regarding, the synthesis of HA in the presence of BPs we developed a new method which allowed to synthesize AL–HA composite nanocrystals with a BPs content up to about 7 wt%. Subsequently, we carried out an in vitro study aimed to investigate the effects of AL incorporation into HA on bone cells response. Osteoblast-like MG63 cells and human osteoclasts were cultured on HA nanocrystals at different AL content. MG63 cells cultured on the composite nanocrystals showed normal morphology, good proliferation and increased values of the differentiation parameters. In particular, when cultured on composites at relatively high AL contents, osteoblasts demonstrated an increased values of alkaline phosphatase activity, collagen type I, and osteocalcin production, as well as significant decrease of matrix metalloproteinases production, with respect both to the control and to pure HA nanocrystals. The presence of AL enhances osteoblast activation and extracellular matrix mineralization processes, without any abnormal collagen degradation. Moreover, the osteoclast number on the composite nanocrystals decreased indicating that the BPs exerts its inhibitory effect on osteoclast proliferation even when incorporated into HA.
These in vitro data on HA nanocrystals synthesized at different extent of Sr and AL indicated that Sr and AL stimulated osteoblast activity and exerted its inhibitory effect on osteoclast proliferation.
These results suggest that Sr-doped HA and AL-doped HA can be usefully employed for the preparation of biomaterials capable to promote osseointegration and bone regeneration, and for local prevention/repair of bone loss.
Based on our in vitro data we carried out an in vivo study in which the experimental biomaterials (HA, SrHA5%, SrHA10%, HA-AL7, HA-AL28) were implanted in lumbar spine, between the transverse processes of L4 and L5 vertebrae, of healthy and osteoporotic female rats. Osteoporosis condition was obtained by the use of ovariectomized animals that were recognized to be a model for estrogen depletion and osteoporosis. Eight weeks after material implantation the bone segment were explanted and analyzed by histological, histomorphometrical and microtomographical analyses. The histological, histomorphometrical and microtomographical tests showed that in healthy bone HA was the best material presenting higher values of bone volume and bone surface when compared with all the other experimental materials. Contrary, in osteoporotic bone HA presented the lower values of bone volume and bone surface when compared with the other materials. In osteoporotic bone HA-AL28 material revealed the higher values of bone volume and bone surface compared with the other experimental materials.
In agreement with our in vitro results, our in vivo model showed that HA-AL 28 was a remarkably effective inhibitor of bone resorption in comparison to HA-Sr5%, HA-Sr10% and HA. Moreover, these results have allowed to detect that HA is an ideal carrier of BPs; HA loaded with appropriate concentration of AL may be ideal for use in spinal fusion of osteoporotic subjects.