Skip to main content
2024 BMES Annual Meeting Main banner
Icon Legend
This session is not in your schedule.
This session is in your schedule. Click again to remove it.
Back

Biomaterials

Biomaterials - Poster Session D

Poster B8 - Optimizing PDGF-BB Tethering on PCL-DCB Scaffolds for Bone Defect Injuries

Friday, October 25, 2024
3:30 PM – 4:30 PM EST
Location: Exhibit Hall E, F & G

Introduction:  Current treatments for critical-sized bone defects fail to effectively restore the native bone microenvironment. Tissue engineering technology has the capacity to improve healing outcomes and overcome the limitations of autologous bone grafts, the current gold standard [1]. One such technology that has shown promise combines biomaterial scaffolds with adipose-derived stem cells (ASCs). Platelet-derived growth factor-BB (PDGF-BB) has been shown to directly enhance osteogenesis of ASCs, which may prove to be useful in aiding regeneration in critical-sized craniofacial bone defects clinically [2]. It has been previously demonstrated in vitro that heparin conjugated decellularized bone (HC-DCB) particles, when seeded within fibrin onto polycaprolactone (PCL) scaffolds along with ASCs, enable a controlled release of growth factors and promote osteogenesis in the presence of PDGF-BB during bone injury healing. However, these enhanced osteogenic effects have not been observed in vivo, which may be caused by resorption of DCB in the early stages of the healing process [3] [4]. To address this limitation, we sought to integrate HC-DCB into the bone scaffold itself. The goal of the project was to develop a method to effectively tether growth factors, including PDGF-BB, via heparin directly to the PCL-DCB scaffold for controlled growth factor delivery.

Materials and

Methods:  DCB Preparation: Decellularized bone was prepared according to a previous protocol [5]. Briefly, calf bones were segmented and washed using high pressure water and decellularization solutions. The bones were then lyophilized, cryomilled, and strained to obtain DCB particles of appropriate size.

Scaffold production: Two-layer PCL sheets were printed using Ultimaker 2+ Connect 3D printer with an 800 um nozzle, at a height of 0.6444 mm, and at approximately 80 °C. The scaffolds were then produced by punching out 4 mm diameter holes using a biopsy punch. The same process was used for creating the PCL-DCB scaffolds except the filament used when printing the sheets was 30% DCB by weight.

Acetone exfoliation: To improve heparin conjugation to PCL-DCB scaffolds, the scaffolds were submerged in acetone for one minute to remove surface-level PCL shielding the DCB without significantly impacting the scaffolds’ structure and mechanical properties. Following exfoliation, the scaffolds were washed in 70% ethanol and then PBS without calcium before air drying overnight.

Heparin conjugation: The PCL-DCB scaffolds that received acetone exfoliation, along with PCL scaffolds dipped in phosphate buffered saline (PBS), PCL-DCB scaffolds dipped in PBS, and DCB powder, were conjugated with heparin by crosslinking DCB particles with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-Hydroxsuccinimide (NHS) [3].

Toluidine Blue Assay: Toluidine Blue Assay was performed on the scaffolds to determine the number of micrograms of heparin per milligram of DCB bound to the scaffolds. The Varioskan LUX Multimode Microplate Reader was used to obtain the results of the assay.

Results, Conclusions, and Discussions: Scanning electron microscope imaging of 30% PCL-DCB scaffolds revealed that a significant amount of DCB appeared to be coated with a thin film of PCL, which would limit heparin conjugation (Figure 1A). Therefore, we developed an acetone exfoliation protocol to remove the top layer of PCL. Using a Toluidine Blue Assay, an increase in glycosaminoglycan (GAG) after heparin conjugation was first validated on free floating DCB particles (Figure 1B). Following acetone exfoliation, the Toluidine Blue assay demonstrated significantly higher mean micrograms of heparin per milligram of DCB for the PCL-DCB scaffolds treated with acetone than the control PCL and PCL-DCB scaffolds treated in PBS (Figure 1C). Based on these results, acetone exfoliation results in a significant increase in the amount of heparin conjugated to the PCL-DCB scaffolds. Thus, future experiments will aim to further validate enhanced DCB surface presentation through SEM imaging and PDGF-BB loading efficiency and release kinetics. Future experiments will also aim to obtain similar results when PCL-DCB scaffolds are exposed to acetone for shorter periods, which is relevant for minimizing acetone’s impact on the scaffolds’ mechanical properties. Next, the impact of acetone exfoliated PCL-DCB scaffolds on ASC osteogenesis will be evaluated in vitro and in vivo.
While a variety of techniques have been used to tether growth factors for delivery, few have been used to tether PDGF [6]. Bone morphogenetic protein-2 (BMP-2), another growth factor with potential in bone regeneration, has more tethering options [6]. However, its clinical use is limited due to necessary high doses in large grafts, which result in negative side effects including ectopic bone formation and infection [3]. Heparin conjugation more appropriate for tethering PDGF than other options, which have been shown to have various issues including an initial burst release of the growth factor [3]. An initial burst release can lead to additional consequences including local or systemic toxicity and short half-life of drugs in vivo [7]. Therefore, creating a PCL-DCB scaffold that can more efficiently conjugate heparin to DCB may enhance osteogenic signaling PDGF to ASCs in vivo for a prolonged period and improve bone regeneration in critical-sized craniofacial defects clinically.

Acknowledgements (Optional):  References
[2] Hung, B. et al. (2015). Platelet-Derived Growth Factor BB Enhances Osteogenesis of Adipose-Derived But Not Bone Marrow-Derived Mesenchymal Stromal/Stem Cells, Stem Cells, 33(9), https://doi.org/10.1002/stem.2060.

[3] Rindone, A. et al. (2019). Heparin-Conjugated Decellularized Bone Particles Promote Enhanced Osteogenic Signaling of PDGF-BB to Adipose-Derived Stem Cells in Tissue Engineered Bone Grafts, Adanced Healthcare Materials, 8(10), https://doi.org/10.1002/adhm.201801565.

[4] Bhumiratana, S. et al. (2016). Tissue-engineered autologous grafts for facial bone reconstruction, Science Translational Medicine, 8(343), https://www.science.org/doi/10.1126/scitranslmed.aad5904.

[5] Hung, B. (2016). Three-Dimensional Printing of Bone Extracellular Matrix for Craniofacial Regeneration, ACS Biometerials Science & Engineering, 2(10), 10.1021/acsbiomaterials.6b00101.

[6] Reed, S., Wu, B. Sustained Growth Factor Delivery in Tissue Engineering Applications. Ann Biomed Eng 42, 1528–1536 (2014). https://doi.org/10.1007/s10439-013-0956-6.

[7] Huang X, Brazel CS (2001). On the importance and mechanisms of burst release in matrix-controlled drug delivery systems, J Control Release, 73(2-3), 10.1016/s0168-3659(01)00248-6.