Research Paper
Influence of structural load-bearing scaffolds on mechanical load- and BMP-2-mediated bone regeneration

https://doi.org/10.1016/j.jmbbm.2016.05.010Get rights and content

Highlights

  • BMP-2/alginate with and without a structural component are assessed in bone defects.

  • Previously, mechanical loads enhanced non-structural hydrogel-mediated bone repair.

  • inclusion of a structural scaffold prevented mechanical load-induced bone regeneration.

  • Bone formed preferentially in regions not containing structural scaffold.

  • Scaffolds with mechanical similarity to native tissue may inhibit load-induced bone healing.

Abstract

A common design constraint in functional tissue engineering is that scaffolds intended for use in load-bearing sites possess similar mechanical properties to the replaced tissue. Here, we tested the hypothesis that in vivo loading would enhance bone morphogenetic protein-2 (BMP-2)-mediated bone regeneration in the presence of a load-bearing PLDL scaffold, whose pores and central core were filled with BMP-2-releasing alginate hydrogel. First, we evaluated the effects of in vivo mechanical loading on bone regeneration in the structural scaffolds. Second, we compared scaffold-mediated bone regeneration, independent of mechanical loading, with alginate hydrogel constructs, without the structural scaffold, that have been shown previously to facilitate in vivo mechanical stimulation of bone formation.

Contrary to our hypothesis, mechanical loading had no effect on bone formation, distribution, or biomechanical properties in structural scaffolds. Independent of loading, the structural scaffolds reduced bone formation compared to non-structural alginate, particularly in regions in which the scaffold was concentrated, resulting in impaired functional regeneration.

This is attributable to a combination of stress shielding by the scaffold and inhibition of cellular infiltration and tissue ingrowth. Collectively, these data question the necessity of scaffold similarity to mature tissue at the time of implantation and emphasize development of an environment conducive to cellular activation of matrix production and ultimate functional regeneration.

Graphical abstract

Introduction

Mechanical stimuli have long been implicated as critical regulators of bone structure and behavior (Wolff, 1892). Mechanical loads control nearly all aspects of bone development, homeostasis, and disease, including load-induced bone modeling and remodeling (Robling and Turner, 2009), disuse-associated osteopenia (Turner et al., 2009), and peri-implant resorption caused by stress shielding (Duyck and Vandamme, 2014). The process of fracture healing is also acutely sensitive to mechanical stimuli, with both the magnitude and mode of interfragmentary motion influencing tissue differentiation, speed of recovery, and ultimate clinical outcome (Kenwright et al., 1986, Röntgen et al., 2010, Wolf et al., 1998).

The consensus of many experimental (Claes et al., 1998, Goodship and Kenwright, 1985, Roux, 1895) and theoretical (Carter et al., 1996, Claes and Heigele, 1999, Lacroix and Prendergast, 2002, Pauwels, 1960, Perren and Cordey, 1980) studies on bone fracture healing demonstrates that cellular lineage specification and tissue differentiation in the fracture callus is controlled by local mechanical conditions, with thresholds and modes of interfragmentary stress and strain regulating callus formation and remodeling. However, the conditions necessary for functional regeneration of critical sized bone defects, which cannot heal without intervention, remain poorly understood and cannot be predicted by the classical theory alone (Claes et al., 1997, Claes et al., 1994, Gómez-Benito et al., 2005).

In recent years, attempts to re-engineer diseased and damaged tissues have demonstrated the importance of both intrinsic and extrinsic mechanical cues for functional regeneration. Intrinsic mechanical cues include inherent properties of the extracellular matrix such as elastic rigidity and viscoelasticity (Chaudhuri et al., 2015, Engler et al., 2006, Huebsch et al., 2015), while extrinsic cues include both static and dynamic forces applied via boundary conditions (Boerckel et al., 2011b, Guldberg et al., 1997). Each of these is of particular importance in bone tissue engineering, where tissue function is fundamentally mechanical. For example, intrinsic matrix mechanical properties are sufficient per se to control lineage specification of stem and progenitor cells (Engler et al., 2006), an observation that has inspired many to pursue controllable, defined matrices for use in tissue engineering, frequently through hydrogels (Huebsch et al., 2015, Kreger et al., 2010, Mason et al., 2013). Similarly, the importance of extrinsic mechanical conditions in tissue formation and adaptation is apparent in the prevalence of dynamic bioreactors for in vitro tissue conditioning and culture (Matziolis et al., 2006, Mauney et al., 2004, Porter et al., 2007). Efforts to control the biomechanical environment in vivo have also revealed profound effects of both intrinsic (Bailey et al., 2011, Huebsch et al., 2015, Jeon et al., 2009) and extrinsic (Boerckel et al., 2011b, Glatt et al., 2012, Roshan-Ghias et al., 2011, Roshan-Ghias et al., 2010) mechanical stimuli on tissue regeneration.

It has long been posited that the ideal biomaterial for tissue engineering would have identical properties to the tissue being replaced (Butler et al., 2009, Butler et al., 2000), balanced by other factors including microstructure, degradation, cell adhesion, and inflammation (Keane and Badylak, 2014). This principle, termed “mechanical similarity,” has been particularly influential in bone tissue engineering, where a common design criterion is to match the properties of native bone, or at least enable physiologic loading without additional stabilization (Butler et al., 2009, Tang et al., 2016).

Recently, we developed a model system to evaluate the role of in vivo mechanical loading on large bone defect regeneration (Boerckel et al., 2009). In this model, a critically sized (8 mm) bone defect is created in the rat femur, requiring treatment to induce healing. To stimulate bone formation, we evaluated delivery of rhBMP-2 using a non-structural alginate hydrogel which released the protein over a time-course of 21 days in vivo (Boerckel et al., 2011a, Kolambkar et al., 2011b). We then tested the effects of in vivo loading and load timing on bone formation, tissue differentiation, and neovascularization by modifying the fixation plates to allow elective actuation of ambulatory load transfer through compliant fixation plates designed to constrain loading to axial compression (Boerckel et al., 2012, Boerckel et al., 2011b, Boerckel et al., 2009). These studies found that limb fixation with the compliant plates implanted in the unlocked configuration at day 0 (i.e. early loading) prevented vascular ingrowth and inhibited bone formation while increasing cartilage formation; however, delaying load initiation to week 4, after onset of bone formation and initiation of defect bridging, significantly enhanced bone formation, biomechanical properties, and local tissue adaptation and remodeling (Boerckel et al., 2012, Boerckel et al., 2011b, Boerckel et al., 2009).

The purpose of the present study was two-fold: first, to evaluate the influence of in vivo mechanical loading on large bone defect regeneration in the presence of a structural load-bearing scaffold capable of supporting and transmitting ambulatory loads to the defect, and second, to compare bone regeneration in the structural and non-structural constructs independent of mechanical stimulation. In Part 1, we tested the hypothesis that mechanical loading would enhance large bone defect regeneration in the presence of a tissue engineering scaffold featuring structural properties and microarchitectural features (i.e. porosity and anisotropy) similar to those of trabecular bone. In Part 2, we compared bone regeneration in the structural scaffolds, independent of mechanical loading, with non-structural hydrogel constructs composed of the same alginate hydrogel but without the structural scaffold support,

Section snippets

Scaffold production

We evaluated two bone tissue-engineering scaffolds selected for their microstructural, mechanical, and functional properties (Fig. 1B). First, mechanically competent, structural scaffolds (PLDL) were fabricated through amorphous co-polymerization of poly(L-lactide) and poly(DL-lactide) with an L:DL ratio of 70:30 (PLDL; Purac America). PLDL pellets were mixed with 10% tricalcium phosphate particles (<200 nm diameter; Sigma Aldrich) and 30% azodicarbonamide (azo; Sigma Aldrich) as a

Part 1: Effects of mechanical loading on BMP-2-mediated bone regeneration in structural PLDL/ALG scaffolds

First, we evaluated the effects of in vivo mechanical loading on large bone defect regeneration in structural, load-bearing scaffolds. Structural PLDL scaffolds were infused, including the cored center region, with alginate hydrogel containing a total of three micrograms rhBMP-2, and implanted in bone defects stabilized by either stiff fixation plates that allowed minimal load sharing, or axially compliant fixation plates, initially implanted in a locked configuration to prevent load transfer,

Discussion

Tissue engineering scaffolds for bone regeneration are frequently designed to match, as closely as possible, the mechanical properties of the native tissue (Butler et al., 2009; Tang et al., 2016). Particularly in bone, structural scaffolds are attractive for load-bearing bone defects to enable load sharing between the construct and fixation hardware to promote functional use as soon as possible. However, if the fixation system is sufficiently durable to withstand in vivo loading, this

Conclusions

Collectively, these observations indicate that structural scaffolds can impede the beneficial effect of mechanical loading by stress shielding and inhibition of cellular infiltration and tissue ingrowth. This suggests that the common design constraint that tissue engineering scaffolds possess similar mechanical properties to the replaced tissue is not requisite for load-bearing tissues, given appropriate fixation conditions. These data instead emphasize the importance of recreating an adequate

Acknowledgments

This work was supported by grants from the Naughton Foundation (to JDB, AMM) and the Indiana Clinical and Translational Science Institute (ICTSI NIH/NCRR UL1TR001108 to JDB). The authors would like to thank Dr. Kenneth Dupont, and Dr. Yash Kolambkar for their assistance, and Dr. David Mooney for providing the RGD alginate.

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