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Molecular design of biologically inspired soft materials for hard tissue regeneration

Final Report Summary - PEPTIDOPAMIN (Molecular design of biologically inspired soft materials for hard tissue regeneration)

Musculoskeletal disorders affects hundreds of millions of people around the world being the most frequent cause of severe long-term pain and physical disability. These conditions place huge burdens on families, societies and health care systems and the problem ted to aggravate due to the increasing world life-expectancy.
Conventional therapy of bone lesions rely on its spontaneous ability to self-heal and remodel. However, there are roughly one million cases of skeletal defects a year that require bone-graft procedures. The shortcomings of current medical grafting procedures to treat severe bone defects continue to drive the development of alternative therapies. Biomaterials started to be envisioned to function as a temporary extracellular matrix (ECM), based on the mechanisms underlying bone regeneration. Such biomaterials should provide a provisional three-dimensional support (scaffold) and interact with cells to control their function, guiding the spatially and temporally complex multicellular processes of tissue formation. These approaches may include both cell-based therapies, where carriers provide structural support for transplanted cells, and acellular therapies, in which materials ideally instruct the physiological milieu towards regeneration by incorporation of biological cues.
Bone ECM itself can be considered a mineralized hydrogel nanocomposite comprising a mineral phase (hydroxyapatite), which represents around 65-70% of the ECM, intimately associated with an organic phase (collagen, proteoglycans, sialoproteins, etc). The organic components of bone give it flexibility and resilience, while the mineral component gives bone its hardness and rigidity. Quantitatively, cells represent a low volume percentage of mature bone. In sharp contrast, osteogenesis is distinguished by high cell contents including abundant osteoprogenitors, while low amount of ECM are present at the early stage of bone formation. In the same way, bone-healing events also recruit a high number of cells.
Due to the above-mentioned features, bone possesses remarkable mechanical properties, perfectly adapted to load-bearing functions. In this sense, scaffolds for bone tissue engineering were often designed to match its mechanical properties, aiming at providing an early mobilization of the injured site and tissue mechanical compatibility. An alternative approach involves the use of an instructive material with lower mechanical properties that is able to provide a proper micro-environment to encapsulated cells, thereby mimicking the regulatory characteristics of natural ECM. If successful, the newly formed bone would then comply with the mechanical requirements.
Hydrogels are attractive materials for tissue engineering because they can homogeneously encapsulate high number of cells, while allowing rapid diffusion of nutrients and metabolites. Hydrogels are capable of retaining large amounts of water, providing a unique 3D environment of great biocompatibility. They can simultaneously serve as scaffolds that provide control drug and protein delivery to tissues and cultures, and serve as adhesives or barriers between tissue and material surfaces. Moreover, they can be designed to be injectable offering several clinical and economical advantages for reconstruction of osseous defects as compared to solid, prefabricated implants. Using flowable materials, complete filling of the defect site can be established by means of minimally invasive techniques, thus avoiding gaps which can lead to fibrous encapsulation or scar formation.
The main aim of this project was to create multifunctional hydrogels designed to be use as injectable systems for minimally invasive surgical procedures in the treatment of bone diseases. Bisphosphonates are a group of well-established drugs widely used for the treatment of bone diseases, including hypercalcemia and osteoporosis. These drugs exhibit a strong affinity to calcium cations as present in bone mineral hydroxyapatite. We hypothesized that covalent attachment of bisphosphonate groups to synthetic, hydrophilic polymers allows for cross-linking of bisphosphonate-grafted hydrogels by means of multivalent cations.
Multi-armed poly(ethyleneglycol) branched molecules with end tethered functional groups were used as a route for immobilization. The 8-armed PEG end-functionalized with bisphosphonate formed self-healing physicaly crosslinked gels in situ when equilibrated with buffer containing Ca2+. Gel elasticity increased with polymer content and calcium concentration, while calcium-mediated crosslinking was shown to be superior over cross-linking mediated by other divalent cations (Zn+2 ; Co+2 ; Ni+2; Sr+2 ;Mn+2; Cu+2; Mg+2).
Moreover, we found that the bisphosphonate after covalently immobilized lost its abilitity to inhibit osteoclastic activity, and did not possess measurable cytotoxicity towards these cells. This is a promising result, since bisphosphonate can be used to create materials without the neither the risk of leaking with consequent local overdose, nor unbalancing uncontrollably bone turnover.
Formed hydrogels are initially moldable and molecularly dynamic, but after incubation in buffer, the hydrogel suffers a hardening process, largely increasing its mechanical strength at the expenses of losing the self-healing properties. We hypothesize that alendronate functionalized multi-arm PEGs can be tuned to match the time dependent mechanical properties of the bone healing process.