Full Length ArticleA biomimetic self-assembling peptide promotes bone regeneration in vivo: A rat cranial defect study
Introduction
There is a major clinical need to replace bone following loss of tissue due to disease (congenital/genetic), ageing or trauma. Autologous bone grafts possess a histocompatibility advantage and are thus the current gold standard for bone replacement. This procedure does however include serious limitations, such as donor site morbidity, limited tissue supply, temporary loss of function and surgical/anaesthetic risks, [1] prompting the development of novel approaches to bone repair based upon tissue regeneration. These have been based upon the use of (either alone or in combinations) adult mesenchymal stem cells [2,3], synthetic/natural scaffolds or cytokines, with the ultimate aim of promoting a tissue rich in collagens and mineral within bone defects [2,[4], [5], [6], [7], [8]]. Biomaterials with hydroxyapatite coatings [9] or materials where all the organic component of allogenic bone has been removed, leaving behind the apatitic mineral phase e.g. Bio-Oss® (Geistlich, Switzerland), OsteoGraf/N (Dentsply, Germany) have proven success in restoring lost bone but can be disadvantaged by their relatively long degradation rates [10]. Bio-Oss® has been reported to be resorbed by osteoclast-like cells in vivo but remnants of the biomaterial have been observed several years post implantation in some patients [[11], [12], [13]]. An ideal biomaterial for bone regeneration should not only provide biocompatibility, appropriate porosity and osteo-conduction/induction but should also possess a biodegradation rate broadly corresponding to that of new bone formation [14].
A range of designer self-assembling peptides (SAPs) are being investigated by various groups for their potential use as scaffolds in regenerative medicine. The ability of SAPs to self-assemble when triggered by environmental cues and their potential to modulate certain parameters such as cell adhesion, mechanical stiffness and biodegradation by varying the peptide sequence makes them an attractive biomaterial for cell-based tissue repair [15]. Aggeli and colleagues designed a family of 11-mer peptides (with varying overall charge, hydrophobicity and polarity) that self-assemble in response to different physico-chemical triggers to produce hydrogels at peptide concentrations of 10–30 mg/mL [16]. P11-4 (primary sequence CH3COQQRFEWEFEQQNH2) is one such SAP with an overall net charge of -2 at physiological pH, that undergoes pH triggered self-assembly to form a self-supporting hydrogel in a concentration dependent manner [[17], [18], [19]]. P11-4 has shown promise as an injectable scaffold for hard tissue engineering applications including its use as an injectable lubricant for osteoarthritis and is already in clinical use as a regenerative treatment for early dental decay (caries) based upon its proven ability to nucleate hydroxyapatite mineral de novo [17,[20], [21], [22]]. Furthermore, P11-4 has a distinct physical advantage over other scaffolds such that when it is shear softened, P11-4 will return to its original gel state, without phase separating [19], offering a capable encapsulating delivery and biomimetic scaffold.
A synergistic combination of an appropriate biomaterial with a suitable cell source is believed to result in regenerated tissue with a biochemical and mechanical composition similar to that of the native tissue [23]. Owing to their intrinsic properties (self-renewal and immunomodulatory), stem cells are a favoured cell source for use in bone tissue regeneration. Whilst bone marrow mesenchymal stem cells (BMMSCs) extracted from the iliac crest remain the best understood and most studied stem cell source used in bone tissue engineering [24,25], human dental pulp stromal cells (HDPSCs) have also been the subject of much investigation since they were first described by Gronthos et al. (2000). They have since been shown to have multiple lineage potential and express many of the cell surface markers used to characterise bone marrow derived MSCs [[26], [27], [28], [29], [30], [31]]. HDPSCs can be easily obtained without any invasive surgery by banking deciduous (“milk”) teeth (“SHED”) [32]. In addition, these cells are documented to possess a higher proliferation rate and enhanced osteogenic differentiation potential when compared to human bone marrow stromal cells (HBMSCs) [33,34]. A combination of these properties makes HDPSCs an attractive cell source for bone tissue engineering.
The aim of this study was to test the hypothesis that HDPSCs used in conjunction with P11-4 hydrogels would enhance regeneration of bone in vivo in a rat calvaria defect model. Our underlying hypothesis was that P11-4's known ability to nucleate hydroxyapatite [20], coupled with the potential of HDPSCs to provide instructional cues to resident cells and their pro-mineralising characteristics, would accelerate bone repair over and above the use of the P11-4 alone.
Section snippets
SAP synthesis
Self-assembling peptide P11-4 is monomeric at high pH and low salt concentration and forms fibrils at low pH and physiological salt concentrations. The peptides used in this study were custom synthesised by CS Bio Co., California and were shown to be >95% (w/w) pure using HPLC analysis. The peptide was sterilised in the dry state using a Gammacell irradiator.
L929 cell culture
L929 cells used for biocompatibility testing experiments were obtained from Sigma Aldrich (UK) and maintained in Dulbecco's Modified Eagle
Biocompatibility testing
Using the MTT assay showed that P11-4 gel extracts had no statistically significant adverse effects upon L929 cell viability (Fig. 1A), suggesting that the P11-4 gel had released no soluble cytotoxic molecules/material. By comparison, the phenol control produced a complete loss of cell viability. Viable L929 cells were observed growing directly in contact with the peptide in the contact cytotoxicity testing (Fig. 1B).
Micro CT analysis: bone volume and bone mineral density
Macroscopically, in samples where defects were filled with P11-4, it was
Discussion
Self-assembling peptides potentially offer advantages over other biomaterials for use in skeletal tissue repair: bulk chemical synthesis ensures economies of scale and higher reproducibility; they have a long shelf life; are free from zoonoses; can be easily and effectively functionalised and can serve to mimic 3D extracellular matrices [23]. The transition from monomeric to self-assembled states of P11-4 can be manipulated according to the environmental conditions required. P11-4 has the
Conclusion
In this study we have demonstrated accelerated healing of calvarial defects as seen by cumulative total bone volume and bone mineral density values, Van Gieson histological staining and osteocalcin and collagen type I immunostaining. This repair was not enhanced by the addition of HDPSCs. Mechanistic factors (including those dictating the micro-environment within the hydrogels) influencing bone repair associated with self-assembling peptides remain to be elucidated, as, indeed, does the fate of
Declaration of Competing Interest
JK is a named co-inventor of the use of self-assembling peptides, including P11-4, as scaffolds for tissue engineering. There are no other conflicts of interest.
Acknowledgements
This work was supported by WELMEC, a Centre of Excellence in Medical Engineering funded by the Wellcome Trust and EPSRC, under grant number WT 088908/Z/09/Z; JK and XY are supported by NIHR through the Leeds Musculoskeletal Biomedical Research Unit. We thank Dr. Amalia Aggeli (Aristotle University of Thessaloniki, Greece), for her advice and input in to the use of self-assembling peptides during this project.
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2022, Dental MaterialsCitation Excerpt :Small osseous islands with blood vessels were also observed. P11-4 previously demonstrated ability to heal critical-size defects in rat calvaria, accelerating the regenerative process, but with some concerns about peptidases reducing SAP potential in cell-laden strategies [46]. Also, high concentrations of P11-4 applied as a gel in periodontal defects did not significantly increase bone height [47].
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2022, BiomaterialsCitation Excerpt :In another study, rat cranial defects (4 mm in diameter) were filled with either organic bone chips or the P11-4 hydrogels for six weeks. Though comparable bone volume at defect site was observed by micro-CT, histological assays showed more extensive new bone formation and higher OCN and COLI levels in the P11-4 group partly due to strong Ca affinity of P11-4 [173]. Similarly, filling SPG178 hydrogels into the bone defects in the medial face of the tibia also displayed increased bone regeneration compared with that of β-TCP [174].