(K-418) Optimizing ASC osteogenesis and viability in spheroid-PCL-DCB scaffolds conjugated with PDGF-tethered heparin

Cranial bone defects drastically decrease patients’ standard of living and are challenging to treat using hard tissue transplantation due to a limited supply of allogenic and autologous donor tissue sources [1] and required immunosuppression [2]. Tissue engineered bone grafts (TEBGs) provide a solution for de novo regenerative bone growth by seeding osteogenic cells onto a structurally supportive scaffold, with the addition of biological growth factors to stimulate cell growth [3]. Adipose-derived stem cells (ASCs) are evidenced to be an effective cell source for bone tissue engineering and demonstrate increased calcium mineralization and proliferation when activated by platelet-derived growth factor BB (PDGF-BB) [1]. ASCs embedded within a fibrin gel seeded onto scaffolds comprised of composite polycaprolactone and decellularized bone matrix (PCL-DCB) show potentiated calcium mineralization [4]. Additionally, simultaneously embedding the fibrin gel with heparin-conjugated DCB particles tethered to PDGF-BB (HC-DCB-PDGF) sustains the delivery of PDGF-BB to ASCs whilst also enhancing osteogenic signaling in vivo [5]. However, these scaffolds lack adequate retention of HC-DCB-PDGF particles due to fibrin degradation [5] and fail to maintain ASC viability in vivo [6]. Therefore, a TEBG that increases retention of PDGF and improves ASC viability is required. ASC spheroids exhibit improved vascularization compared to monodispersed ASCs, and thus provide a strategy for increasing ASC viability within TEBGs [7]. Furthermore, conjugating PDGF-bound heparin directly to DCB within PCL-DCB scaffolds may mitigate undesirable diffusion. We aim to engineer a TEBG that incorporates ASC spheroids seeded onto HC-PCL-DCB-PDGF scaffolds to enhance ASC viability in vitro.

ASC expansion. ASCs were expanded for one passage in an expansion growth media consisting of high-glucose DMEM (Gibco Invitrogen), 10% fetal bovine serum (FBS; Atlanta Biologicals), 1% penicillin/streptomycin (GIBCO Invitrogen), and 1 ng/mL FGF-2 (PeproTech). ASCs underwent an additional passage before experimentation.

Scaffold construction. PCL-only scaffolds were produced by printing two-layer PCL sheets at 0.644 mm height and punching out scaffolds for a 4mm diameter. PCL-DCB scaffolds with 45% bone-by-mass were printed after filament extrusion [4].

ASC spheroid formation. ASCs were collected via the hanging drop method [7] to produce spheroids containing 8,000 cells each.

ASC aggregate formation. ASCs were dispersed onto a petri dish covered in agarose and equilibrated with expansion media. Cells were incubated overnight for aggregation and were then collected and suspended in expansion media.

Scaffold seeding. ASCs were resuspended in fibrinogen (8 mg/mL final; Sigma) and added to thrombin (2 U/mL final; Sigma) before being seeded onto PCL-only scaffolds. 30,000 cells were added per scaffold.

Osteogenic media preparation. Osteogenic media consisted of low-glucose DMEM (Gibco Invitrogen), 6% FBS, 1% penicillin/streptomycin, 10 mM β-glycerophosphate (Sigma) and 50 μM L-ascorbic acid-2-phosphate.

ASC 3D culture. ASCs were cultured in scaffolds for 21 days in expansion or osteogenic media. Media was replaced every other day.

Assays. Calcium content was quantified via Calcium LiquiColor Test (Stanbio). DNA content was quantified using a PicoGreen dsDNA quantitation kit (Molecular Probes). Alizarin Red staining (Sigma) was performed directly on scaffolds.

Results

In vitro osteogenesis. ASCs were seeded onto PCL-only scaffolds as either a monodispersed layer, aggregates, or spheroids and cultured for 21 days. Results after 21 days show that calcium mineralization per DNA (ug per ug) per scaffold was highest in scaffolds seeded with spheroids (Figure 1C), demonstrating the ability of spheroids to maintain osteogenesis. Scaffolds treated with aggregates and monodispersed ASCs showed similar levels of calcium mineralization per DNA. These results were corroborated by Alizarin Red staining (Figure 2).

PCL-DCB scaffold fabrication. 45% bone-by-mass PCL-DCB scaffolds were fabricated with varied printer nozzle diameters and temperatures (Figure 3), with a 155° Celsius nozzle demonstrating the best print. 50% and 70% bone-by-mass PCL-DCB scaffolds were unable to be printed.

Conclusions

Spheroids and aggregates suggest an increased capability for calcium mineralization (Figure 1) as compared to monodispersed ASCs. Considering especially evidence suggesting spheroid cultures increase cell viability [8,9], spheroids can be considered a viable option for improving osteogenesis of ASCs in scaffold cultures in vitro. Spheroids provide the ability to increase calcium mineralization without increasing cell count.

While the 45% bone-by-mass PCL-DCB scaffold was able to be printed at 155° Celsius, the filament and scaffolds were brittle and thin. Both the printer conditions and the concentration of bone-by-mass require further optimization to ensure mechanical strength and heparin binding.

Discussion & Future Results

In vitro ASC viability. Future experiments will test the ability of spheroids to increase proliferation and osteogenesis when supplemented with PDGF-BB. Furthermore, measurement of the activation of the PI3-Akt pathway, which plays an important role in cell proliferation [10], will be incorporated to demonstrate increased viability of ASCs in spheroid form.

HC-PCL-DCB-PDGF scaffolds. After bone-by-mass concentration of PCL-DCB scaffolds is optimized, heparin will be conjugated to the PCL-DCB scaffolds. Conjugation efficiency will be measured via a toluidine blue assay. PDGF will then be loaded onto the scaffold and both loading efficiency and PDGF release will be analyzed. ASCs will then be cultured on HC-PCL-DCB-PDGF scaffolds.

[1] Hung, B. P., Hutton, D. L., Kozielski, K. L., Bishop, C. J., Naved, B., Green, J. J., Caplan, A. I., Gimble, J. M., Dorafshar, A. H., & Grayson, W. L. (2015). Platelet-derived growth factor BB enhances osteogenesis of adipose-derived but not bone marrow-derived mesenchymal stromal/stem cells. Stem Cells, 33(9), 2773–2784. https://doi.org/10.1002/stem.2060

[2] Szpalski, C., Barr, J., Wetterau, M., Saadeh, P. B., & Warren, S. M. (2010). Cranial bone defects: Current and future strategies. Neurosurgical Focus, 29(6). https://doi.org/10.3171/2010.9.focus10201

[3] Chan, B. P., & Leong, K. W. (2008). Scaffolding in tissue engineering: General approaches and tissue-specific considerations. European Spine Journal, 17(S4), 467–479. https://doi.org/10.1007/s00586-008-0745-3

[4] Cheng, N.-C., Chen, S.-Y., Li, J., & Young, T.-H. (2013). Short-Term Spheroid Formation Enhances the Regenerative Capacity of Adipose-Derived Stem Cells by Promoting Stemness, Angiogenesis, and Chemotaxis. Stem Cells Translational Medicine, 2(8), 584–594. https://doi.org/10.5966/sctm.2013-0007

[5] Frederik Penzien Mamsen, Munthe-Fog, L., Mungal, K., Duscher, D., Mikkel Taudorf, Katz, A. J., & Stig-Frederik Trojahn Kølle. (2021). Differences of embedding adipose-derived stromal cells in natural and synthetic scaffolds for dermal and subcutaneous delivery. Stem Cell Research & Therapy, 12(1). https://doi.org/10.1186/s13287-020-02132-5

[6] Hemmings, B. A., & Restuccia, D. F. (2012). PI3K-PKB/Akt Pathway. Cold Spring Harbor Perspectives in Biology, 4(9), a011189–a011189. https://doi.org/10.1101/cshperspect.a011189

[7] Hung, B. P., Naved, B. A., Nyberg, E. L., Dias, M., Holmes, C. A., Elisseeff, J. H., Dorafshar, A. H., & Grayson, W. L. (2016). Three-Dimensional Printing of Bone Extracellular Matrix for Craniofacial Regeneration. ACS Biomaterials Science & Engineering, 2(10), 1806–1816. https://doi.org/10.1021/acsbiomaterials.6b00101

[8] Hutton, D. L., Moore, E. M., Gimble, J. M., & Grayson, W. L. (2013). Platelet-Derived Growth Factor and Spatiotemporal Cues Induce Development of Vascularized Bone Tissue by Adipose-Derived Stem Cells. Tissue Engineering Part A, 19(17-18), 2076–2086. https://doi.org/10.1089/ten.tea.2012.0752

[9] Luo, L., Zhang, W., Wang, J., Zhao, M., Shen, K., Jia, Y., Li, Y., Zhang, J., Cai, W., Xiao, D., Bai, X., Liu, K., Wang, K., Zhang, Y., Zhu, H., Zhou, Q., & Hu, D. (2021). A Novel 3D Culture Model of Human ASCs Reduces Cell Death in Spheroid Cores and Maintains Inner Cell Proliferation Compared With a Nonadherent 3D Culture. Frontiers in Cell and Developmental Biology, 9. https://doi.org/10.3389/fcell.2021.737275

[10] Rindone, A. N., Kachniarz, B., Achebe, C. C., Riddle, R. C., O’Sullivan, A. N., Dorafshar, A. H., & Grayson, W. L. (2019). Craniofacial Bone‐Tissue Engineering: Heparin‐Conjugated Decellularized Bone Particles Promote Enhanced Osteogenic Signaling of PDGF‐BB to Adipose‐Derived Stem Cells in Tissue Engineered Bone Grafts (Adv. Healthcare Mater. 10/2019). Advanced Healthcare Materials, 8(10), 1970042. https://doi.org/10.1002/adhm.201970042