Semaphorin 3A Delivered by a Rapidly Polymerizing Click Hydrogel Overcomes Impaired Implant Osseointegration in a Rat Type 2 Diabetes Model

Dental implants have enabled the restoration of dentition in the growing aging population, which has resulted in improved health for many individuals. However, poor bone quality associated with diseases like type 2 diabetes mellitus (T2DM) and osteoporosis impacts implant survival [1,2]. Even though some reports have indicated that the success rate of dental implants in diabetic patients is comparable to non-diabetic patients, current evidence supports the hypothesis that T2DM patients with a poorly or moderately controlled glycemic level have a higher early implant failure rate than well-controlled T2DM patients [3], [4], [5], [6], [7], [8], [9].

Implant success depends on osseointegration [10, 11]. Our group has previously demonstrated that surface treatments that produce macro/micro/nano-textured features on titanium implants can enhance osseointegration [10, 12, 13]. A contributing factor to this is the production of osteogenic factors by bone marrow stromal cells (BMSCs) on the surface, including bone morphogenetic protein-2 (BMP2) and BMP4, as well as the downregulation of inflammatory mediators like interleukin 6 (IL6) [14], [15], [16].

Recently, we found that the production of the nerve-derived protein semaphorin 3A (sema3A) by BMSCs was sensitive to Ti surface properties, including microtopography and chemistry. Sema3A is a known osteoprotective factor that both increases bone formation and inhibits bone resorption [17], [18], [19], [20], [21]. It has been reported to promote osteoblast differentiation of BMSCs isolated from diabetic rats [22]. Furthermore, sema3A was shown to mitigate the inhibitory effect of high glucose on osteogenesis using a mouse osteoblast cell line in vitro [23]. These studies, together with our observations on the effects of sema3A on enhancing BMSC differentiation on microtextured titanium surfaces [24] suggested that sema3A might be a useful therapeutic for promoting peri-implant osteogenesis in T2DM in vivo. We hypothesized that localized delivery of sema3A at the site of implantation would be a more powerful therapeutic strategy to impact surrounding osteoblast-lineage cells, mobilizing them to enhance osseointegration under compromised conditions, such as T2DM.

One of the challenges when administering this potent factor as a potential therapeutic is delivering it at an adequate concentration within a sufficient therapeutic window to elicit the desired cellular effects and achieve a favorable outcome in compromised patients. Our strategy for achieving sustained delivery of sema3A was to use a minimally invasive, rapidly polymerizing, click chemistry-based hydrogel. Hydrogels are biomaterials composed of hydrated, crosslinked networks of water-soluble polymers that have been extensively employed as vehicles for drug encapsulation and sustained delivery of therapeutics. The bio-orthogonal, injectable hydrogel described in this study forms a stable gel in under 90 s at 37°C through a ring-strain promoted Cu-free click reaction between azide functionalized PEG polymers and a DBCO functionalized PEG crosslinker without external photo-initiators and without generating heat. The rapid encapsulation of bioactive molecules into the hydrogel maintains the biologics at the delivery site over an extended time. Ester linkages degrade in vivo and ultimately release the loaded molecules [25]. When biocompatibility of a similar click hydrogel was tested in critical size defects in the craniums of weanling and adult mice, degradation occurred over 5 to 10 days without releasing any toxic molecules or by-products. Histologically, there was no evidence of pathology in the surrounding tissues [25,26] suggesting that the current formulation would be an ideal platform for sema3A delivery.

In these studies, we employed the Zucker Diabetic Sprague Dawley (ZDSD) rats, a clinically relevant T2DM rodent model. Unlike the prevailing diabetic rodent models that are either induced by chemical toxin injection (such as streptozotocin or alloxan) [27,28] or via genetic mutations (leptin or leptin receptor deficiency) [29], ZDSD rats develop T2DM spontaneously when fed a high fat diet. Thus, the ZDSD rat is a model that closely resembles factors contributing to the manifestation of T2DM in humans [30], [31], [32]. Using this model will allow clinicians to better translate findings to benefit the human condition and improve clinical outcomes when managing T2DM patients receiving implants.

The goal of this study was to determine if sema3A delivered locally via the click hydrogel could impact osteogenesis in a compromised bone model. The formulation selected for this initial study was shown to be effective for delivery of peptides, antibodies, and drugs in our previous studies, although the hydrogel characteristics can be modified to vary the concentration of factor within the gel, the gel degradation kinetics, and the kinetics of factor release. Here we report the impact of T2DM on the osseointegration of microtextured titanium implants in the ZDSD rat model and deliver sema3A in two modalities: via direct incorporation into the click hydrogel and via local injection of sema3A over the implant site. In addition, the effect of sustained delivery versus burst release of sema3A is demonstrated in this study. Finally, we assessed the effects of T2DM on the ability of rat calvarial osteoblasts to respond to the Ti surface in vitro.

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