Sander C. G. Leeuwenburgh

Organic-inorganic surface modifications for titanium implant surfaces.

This paper reviews current physicochemical and biochemical coating techniques that are investigated to enhance bone regeneration at the interface of titanium implant materials. By applying coatings onto titanium surfaces that mimic the organic and inorganic components of living bone tissue, a physiological transition between the non-physiological titanium surface and surrounding bone tissue can be established. In this way, the coated titanium implants stimulate bone formation from the implant surface, thereby enhancing early and strong fixation of bone-substituting implants. As such, a continuous transition from bone tissue to implant surface is induced. This review presents an overview of various techniques that can be used to this end, and that are inspired by either inorganic (calcium phosphate) or organic (extracellular matrix components, growth factors, enzymes, etc.) components of natural bone tissue. The combination, however, of both organic and inorganic constituents is expected to result into truly bone-resembling coatings, and as such to a new generation of surface-modified titanium implants with improved functionality and biological efficacy.

Combined delivery of BMP-2 and bFGF from nanostructured colloidal gelatin gels and its effect on bone regeneration in vivo

During the process of bone regeneration, a multitude of morphogenetic signaling factors regulate cellular behavior and ultimately tissue response. These factors are presented to cells under strong spatial and temporal control, which stresses the relevance of controlled delivery of multiple growth factors for bone tissue regeneration. This demand for biomimetic delivery has prompted the development of a novel generation of biomaterials that is capable of delivering multiple growth factors in a controlled manner. Therefore, the current study has exploited the strong capacity of colloidal gels solely made of oppositely charged gelatin nanospheres to obtain controlled release of angiogenic and osteogenic growth factors. The release kinetics of dual delivery of osteogenic bone morphogenetic protein-2 (BMP-2) and angiogenic basic fibroblast growth factor (bFGF) were investigated in vitro by radiolabeling the respective growth factors and monitoring their release in vitro. Furthermore, the effect of single or dual delivery of BMP-2 and bFGF on bone regeneration was evaluated in vivo using a rat femoral condyle defect model. The in vitro results confirmed that the delivery kinetics of BMP-2 and/or bFGF are more dependent on the degree of crosslinking than on the type of gelatin. Sequential release characterized by rapid release of angiogenic bFGF and more sustained release of BMP-2 was obtained by loading bFGF onto cationic nanospheres of low crosslinking density and BMP-2 onto anionic nanospheres of high crosslinking density. The in vivo study demonstrated the biocompatibility and biodegradability of bare colloidal gelatin gels, and did not show any adverse effects on the process of bone healing after 4 week of implantation since the volumes of new bone formation were comparable to empty control defects. An obvious stimulatory effect on bone regeneration was observed for the colloidal gels loaded with BMP-2, whereas bFGF-loaded colloidal gelatin gels did not influence the rate of bone regeneration. In contrast, the combined delivery of BMP-2 and bFGF resulted into an inhibitory effect on osteogenesis under the current experimental conditions. Summarizing, the current study proved that nanostructured colloidal gelatin gels are suitable carriers for programmed and sustained release of multiple therapeutic proteins for tissue regeneration.

Sustained release of BMP-2 in bioprinted alginate for osteogenicity in mice and rats

The design of bioactive three-dimensional (3D) scaffolds is a major focus in bone tissue engineering. Incorporation of growth factors into bioprinted scaffolds offers many new possibilities regarding both biological and architectural properties of the scaffolds. This study investigates whether the sustained release of bone morphogenetic protein 2 (BMP-2) influences osteogenicity of tissue engineered bioprinted constructs. BMP-2 loaded on gelatin microparticles (GMPs) was used as a sustained release system, which was dispersed in hydrogel-based constructs and compared to direct inclusion of BMP-2 in alginate or control GMPs. The constructs were supplemented with goat multipotent stromal cells (gMSCs) and biphasic calcium phosphate to study osteogenic differentiation and bone formation respectively. BMP-2 release kinetics and bioactivity showed continuous release for three weeks coinciding with osteogenicity. Osteogenic differentiation and bone formation of bioprinted GMP containing constructs were investigated after subcutaneous implantation in mice or rats. BMP-2 significantly increased bone formation, which was not influenced by the release timing. We showed that 3D printing of controlled release particles is feasible and that the released BMP-2 directs osteogenic differentiation in vitro and in vivo.

Bone response to fast-degrading, injectable calcium phosphate cements containing PLGA microparticles

Apatitic calcium phosphate cements (CPC) are frequently used to fill bone defects due to their favourable clinical handling and excellent bone response, but their lack of degradability inhibits complete bone regeneration. In order to render these injectable CaP cements biodegradable, hollow microspheres made of poly (D,L-lactic-co-glycolic) acid (PLGA) have been previously used as porogen since these microspheres were shown to be able to induce macroporosity upon degradation as well as to accelerate CPC degradation by release of acid degradation products. Recently, the capacity of PLGA microspheres to form porosity in situ in injectable CPCs was optimized by investigating the influence of PLGA characteristics such as microsphere morphology (dense vs. hollow) and end-group functionalization (acid terminated vs. end-capped) on acid production and corresponding porosity formation in vitro. The current study has investigated the in vivo bone response to CPCs containing two types of microspheres (hollow and dense) made of PLGA with two different end-group functionalizations (end capped and acid terminated). Microspheres were embedded in CPC and injected in the distal femoral condyle of New Zealand White Rabbits for 6 and 12 weeks. Histological results confirmed the excellent biocompatibility and osteoconductivity of all tested materials. Composites containing acid terminated PLGA microspheres displayed considerable porosity and concomitant bone ingrowth after 6 weeks, whereas end capped microspheres only revealed open porosity after 12 weeks of implantation. In addition, it was found that dense PLGA microspheres induced significantly more CPC degradation and bone tissue formation compared to hollow PLGA microspheres. In conclusion, it was shown that PLGA microspheres have a strong capacity to induce fast degradation of injectable CPC and concomitant replacement by bone tissue by controlled release of acid polymeric degradation products without compromising the excellent biocompatibility and osteoconductivity of the CPC matrix.

The osteogenic effect of electrosprayed nanoscale collagen/calcium phosphate coatings on titanium.

For orthopedic and dental implants, the ultimate goal is to obtain a life-long secure anchoring of the implant in the native surrounding bone. To this end, nanoscale calcium phosphate (CaP) and collagen-CaP (col-CaP) composite coatings have been successfully deposited using the electrospray deposition (ESD) technique. In order to study to what extent the thickness of these coatings can be reduced without losing coating osteogenic properties, we have characterized the mechanical and biological coating properties using tape tests (ASTM D-3359) and in vitro cell culture experiments, respectively. Co-deposition of collagen significantly improved coating adhesive and cohesive strength, resulting in a remarkably high coating retention of up to 97% for coating thicknesses below 100 nm. In vitro cell culture experiments showed that electrosprayed CaP and col-CaP composite coatings enhanced osteoblast differentiation, leading to improved mineral deposition. This effect was most pronounced upon co-deposition of collagen with CaP, and these coatings displayed osteogenic effects even for a coating thickness of below 100 nm.

Comparison of micro- vs. nanostructured colloidal gelatin gels for sustained delivery of osteogenic proteins: Bone morphogenetic protein-2 and alkaline phosphatase

Colloidal gels have recently emerged as a promising new class of materials for regenerative medicine by employing micro- and nanospheres as building blocks to assemble into integral scaffolds. To this end, physically crosslinked particulate networks are formed that are injectable yet cohesive. By varying the physicochemical properties of different particle populations, the suitability of colloidal gels for programmed delivery of multiple therapeutic proteins is superior over conventional monolithic gels that lack this strong capacity for controlled drug release. Colloidal gels made of biodegradable polymer micro- or nanospheres have been widely investigated over the past few years, but a direct comparison between micro- vs. nanostructured colloidal gels has not been made yet. Therefore, the current study has compared the viscoelastic properties and capacity for drug release of colloidal gels made of oppositely charged gelatin microspheres vs. nanospheres. Viscoelastic properties of the colloidal gelatin gels were characterized by rheology and simple injectability tests, and in vitro release of two selected osteogenic proteins (i.e. bone morphogenetic protein-2 (BMP-2) and alkaline phosphatase (ALP)) from the colloidal gelatin gels was evaluated using radiolabeled BMP-2 and ALP. Nanostructured colloidal gelatin gels displayed superior viscoelastic properties over microsphere-based gels in terms of elasticity, injectability, structural integrity, and self-healing behavior upon severe network destruction. In contrast, microstructured colloidal gelatin gels exhibited poor gel strength and integrity, unfavorable injectability, and did not recover after shearing, resulting from the poor gel cohesion due to insufficiently strong interparticle forces. Regarding the capacity for drug delivery, sustained growth factor (BMP-2) release was obtained for both micro- and nanosphere-based gels, the kinetics of which were mainly depending on the particle size of gelatin spheres with the same crosslinking density. Therefore, the optimal gelatin carrier for drug delivery in terms of particle size and crosslinking density still needs to be established for specific clinical indications that require either short-term or long-term release. It can be concluded that nanostructured colloidal gelatin gels show great potential for sustained delivery of therapeutic proteins, whereas microstructured colloidal gelatin gels are not sufficiently cohesive as injectables for biomedical applications.

Oppositely charged gelatin nanospheres as building blocks for injectable and biodegradable gels

www.MaterialsViews.com C O M M Oppositely Charged Gelatin Nanospheres as Building Blocks for Injectable and Biodegradable Gels U N IC A T Huanan Wang , Morten B. Hansen , Dennis W. P. M. Lowik , Jan C. M. van Hest , Yubao Li , John A. Jansen , and Sander C. G. Leeuwenburgh * IO N The emergence of regenerative medicine has led to a paradigm shift in the design of novel biomaterials, which are now increasingly considered as (bio)active scaffolds that induce tissue regeneration as opposed to the more traditional concept of passively accepted implant materials. [ 1 ] In order to present biological stimuli to the physiological environment and to trigger tissue repair, optimal integration of synthetic biomaterials within the surrounding tissue is of paramount importance. In that respect, hydrogels made from biodegradable polymers are ideal candidates, since they are generally biocompatible, biodegradable, and, in some cases, injectable. [ 2–4 ] In addition, polymeric hydrogels can act as a reservoir for sustained release of therapeutic and signaling agents. [ 4 ] Nevertheless, current gelbased materials exhibit a rather poor ability to present multiple signaling molecules at programmed time-points and release rates. Colloidal gels, on the other hand, have recently been identifi ed as a promising “bottom-up” strategy for the design of highly functional scaffolds by employing microor nanometer-scale particles as building blocks to assemble into shape-specifi c bulk materials. [ 5–13 ] To this end, interparticle interactions such as electrostatic forces, [ 14 ] magnetic forces, [ 14 ] hydrophobic interactions [ 15 ] and steric hindrance [ 16 ] can be exploited to induce selfassembly of microor nanoparticles into integrated scaffolds. By incorporation of bioactive agents (e.g., enzymes, growth factors and/or biomineral nanocrystals) into these particulate building blocks of variable biodegradability, injectable gels with micrometer-scale resolution and complexity can be formed. This new class of materials would offer virtually unlimited degree of freedom with respect to their capacity for programmed drug release of multiple biomolecules at predetermined release rates. Charged polymeric microor nanospheres are the most obvious building blocks for the design of such injectable and

In vitro degradation rate of apatitic calcium phosphate cement with incorporated PLGA microspheres

Calcium phosphate cements (CPCs) are frequently used as bone substitute material. Despite their superior clinical handling and excellent biocompatibility, they exhibit poor degradability, which limits bone ingrowth into the implant. Microspheres were prepared from poly(d,l-lactic-co-glycolic acid) (PLGA) and included in injectable CPCs as porogens in order to enhance its macroporosity after the polymeric microspheres had degraded. Upon degradation of the PLGA microspheres, acid is produced that enhances the dissolution rate of the CPC. However, the effect of the characteristics of PLGA microspheres on the degradation rate of CPCs has never been studied before. Therefore, the purpose of the current study was to investigate the dependence of CPC degradation on the chemical and morphological characteristics of incorporated PLGA microspheres. With respect to the chemical characteristics of the PLGA microspheres, the effects of both PLGA molecular weight (5, 17 and 44kDa) and end-group functionalization (acid-terminated or end-capped) were studied. In addition, two types of PLGA microspheres, differing in morphology (hollow vs. dense), were tested. The results revealed that, although both chemical parameters clearly affected the polymer degradation rate when embedded as hollow microspheres in CPC, the PLGA and CPC degradation rates were mainly dependent on the end-group functionalization. Moreover, it was concluded that dense microspheres were more efficient porogens than hollow ones by increasing the CPC macroporosity during in vitro incubation. By combining all test parameters, it was concluded that dense PLGA microspheres consisting of acid-terminated PLGA of 17kDa exhibited the highest and fastest acid-producing capacity and correspondingly the highest and fastest amount of porosity. In conclusion, the data presented here indicate that the combination of dense, acid-terminated PLGA microspheres with CPC emerges as a successful combination to achieve enhanced apatitic CPC degradation.

Development of bone substitute materials: from ‘biocompatible’ to ‘instructive’

Progress made in basic research in the last decades led to a tremendous increase in quality of clinically applied bone substitute materials (polymers, ceramics and composites). The desired biological performance of these materials has consequently shifted from a passive role where materials were merely accepted by the body to an active role in which materials instruct their biological surroundings. Bone substitute materials were traditionally based on bioceramics, that can be optimized in terms of composition, structure and porosity. Now, polymers are increasingly gaining importance for use in medical applications due to their high versatility. This review provides an overview of the evolution from 1st generation biotolerant and bioinert materials via 2nd generation bioresponsive bone substitutes towards 3rd generation bioinstructive bone substitute materials that possess inherent biological cues for bone regeneration.

Self-healing hybrid nanocomposites consisting of bisphosphonated hyaluronan and calcium phosphate nanoparticles

Non-covalent interactions are often regarded as insufficient to construct macroscopic materials of substantial integrity and cohesion. However, the low binding energy of such reversible interactions can be compensated by increasing their number to work in concert to create strong materials. Here we present the successful development of an injectable, cohesive nanocomposite hydrogel based on reversible bonds between calcium phosphate nanoparticles and bisphosphonate-functionalized hyaluronic acid. These nanocomposites display a capacity for self-healing as well as adhesiveness to mineral surfaces such as enamel and hydroxyapatite. Most importantly, these non-covalently cross-linked composites are surprisingly robust yet biodegradable upon extensive in vitro and in vivo testing and show bone interactive capacity evidenced by bone ingrowth into material remnants. The herein presented method provides a new methodology for constructing nanoscale composites for biomedical applications, which owe their integrity to reversible bonds.