Insufficient vascularization is the main barrier to creating engineered bone grafts for treating large and ischemic defects. Modular tissue engineering approaches have promise in this application because of the ability to combine tissue types and localize microenvironmental cues to drive desired cell function. In direct bone formation approaches, it is challenging to maintain sustained osteogenic activity, since vasculogenic cues can inhibit tissue mineralization. This study harnessed the physiological process of endochondral ossification to create multiphase tissues that allowed concomitant mineralization and vessel formation. Mesenchymal stromal cells in pellet culture were differentiated toward a cartilage phenotype, followed by induction to chondrocyte hypertrophy. Hypertrophic pellets (HPs) exhibited increased alkaline phosphatase activity, calcium deposition, and osteogenic gene expression relative to chondrogenic pellets. In addition, HPs secreted and sequestered angiogenic factors, and supported new blood vessel formation by cocultured endothelial cells and undifferentiated stromal cells. Multiphase constructs created by combining HPs and vascularizing microtissues and maintained in an unsupplemented basal culture medium were shown to support robust vascularization and sustained tissue mineralization. These results demonstrate a promising in vitro strategy to produce multiphase-engineered constructs that concomitantly support the generation of mineralized and vascularized tissue in the absence of exogenous osteogenic or vasculogenic medium supplements.
Impact Statement
Healing of large and complex bone fractures requires the regeneration of both bone tissue and an associated blood supply to allow the development and maturation of the new tissue. This work harnesses the natural process of endochondral ossification (bone development from a cartilage precursor) to create multiphase tissues that can develop into vascularized bone. It therefore presents a promising approach to creating advanced bone grafts that can be used to treat challenging orthopedic defects.
NovoselEC, KleinhansC, KlugerPJ. Vascularization is the key challenge in tissue engineering. Adv Drug Deliv Rev, 2011; 63(4–5):300–311; doi: 10.1016/j.addr.2011.03.004
2.
SantosMI, ReisRL. Vascularization in bone tissue engineering: Physiology, current strategies, major hurdles and future challenges. Macromol Biosci, 2010; 10(1):12–27; doi: 10.1002/mabi.200900107
3.
FanJ, BiL, JinD, et al.Microsurgical techniques used to construct the vascularized and neurotized tissue engineered bone. Biomed Res Int, 2014; 2014:281872; doi: 10.1155/2014/281872
4.
LiuH, ChenH, HanQ, et al.Recent advancement in vascularized tissue-engineered bone based on materials design and modification. Mater Today Bio, 2023; 23:100858; doi: 10.1016/j.mtbio.2023.100858
5.
WenzA, TjoengI, SchneiderI, et al.Improved vasculogenesis and bone matrix formation through coculture of endothelial cells and stem cells in tissue-specific methacryloyl gelatin-based hydrogels. Biotechnol Bioeng, 2018; 115(10):2643–2653; doi: 10.1002/bit.26792
6.
ChiesaI, MariaCD, LapomardaA, et al.Endothelial cells support osteogenesis in an in vitro vascularized bone model developed by 3D bioprinting. Biofabrication, 2020; 12(2):025013; doi: 10.1088/1758-5090/ab6a1d
7.
HuttonDL, MooreEM, GimbleJM, et al.Platelet-derived growth factor and spatiotemporal cues induce development of vascularized bone tissue by adipose-derived stem cells. Tissue Eng Part A, 2013; 19(17–18):2076–2086; doi: 10.1089/ten.tea.2012.0752
8.
CorreiaC, GraysonW, EtonR, et al.Human adipose-derived cells can serve as a single-cell source for the in vitro cultivation of vascularized bone grafts. J Tissue Eng Regen Med, 2014; 8(8):629–639; doi: 10.1002/term
9.
KolbeM, XiangZ, DohleE, et al.Paracrine effects influenced by cell culture medium and consequences on microvessel-like structures in cocultures of mesenchymal stem cells and outgrowth endothelial cells. Tissue Eng Part A, 2011; 17(17–18):2199–2212; doi: 10.1089/ten.tea.2010.0474
10.
ChaiM, GuC, ShenQ, et al.Hypoxia alleviates dexamethasone-induced inhibition of angiogenesis in cocultures of HUVECs and rBMSCs via HIF-1α. Stem Cell Res Ther, 2020; 11(1):343; doi: 10.1186/s13287-020-01853-x
11.
SchottNG, StegemannJP. Coculture of endothelial and stromal cells to promote concurrent osteogenesis and vasculogenesis. Tissue Eng Part A, 2021; 27(21–22):1376–1386; doi: 10.1089/ten.tea.2020.0330
12.
AlmubarakS, NethercottH, FreebergM, et al.Tissue engineering strategies for promoting vascularized bone regeneration. Bone, 2016; 83:197–209; doi: 10.1016/J.BONE.2015.11.011
13.
FriendNE, RiojaAY, KongYP, et al.Injectable pre-cultured tissue modules catalyze the formation of extensive functional microvasculature in vivo. Sci Rep, 2020; 10(1):15562; doi: 10.1038/s41598-020-72576-5
14.
MishraR, RouxBM, PosukonisM, et al.Effect of prevascularization on in vivo vascularization of poly(propylene fumarate)/fibrin scaffolds. Biomaterials, 2016; 77:255–266; doi: 10.1016/j.biomaterials.2015.10.026
15.
PerryL, MerdlerU, ElishaevM, et al.Enhanced host neovascularization of prevascularized engineered muscle following transplantation into immunocompetent versus immunocompromised mice. Cells, 2019; 8(12).
16.
RouxBM, AkarB, ZhouW, et al.Preformed vascular networks survive and enhance vascularization in critical sized cranial defects. Tissue Eng Part A, 2018; 24(21–22):1603–1615; doi: 10.1089/ten.tea.2017.0493
17.
AnnamalaiRT, HongX, SchottNG, et al.Injectable osteogenic microtissues containing mesenchymal stromal cells conformally fill and repair critical-size defects. Biomaterials, 2019; 208:32–44; doi: 10.1016/j.biomaterials.2019.04.001
18.
OryanA, KamaliA, MoshiriA, et al.Role of mesenchymal stem cells in bone regenerative medicine : what is the evidence? Cells Tissues Organs, 2017; 204(2):59–83; doi: 10.1159/000469704
19.
SchottNG, FriendNE, StegemannJP. Coupling osteogenesis and vasculogenesis in engineered orthopedic tissues. Tissue Eng Part B Rev, 2021; 27(3):199–214; doi: 10.1089/ten.teb.2020.0132
20.
ForrestalDP, KleinTJ, WoodruffMA. Challenges in engineering large customized bone constructs. Biotechnol Bioeng, 2017; 114(6):1129–1139; doi: 10.1002/bit.26222
21.
SearsV, GhoshG. Harnessing mesenchymal stem cell secretome: Effect of extracellular matrices on pro-angiogenic signaling. Biotechnol Bioeng, 2020; 117(4):1159–1171; doi: 10.1002/bit.27272
22.
RohringerS, HofbauerP, SchneiderKH, et al.Mechanisms of vasculogenesis in 3D fibrin matrices mediated by the interaction of adipose-derived stem cells and endothelial cells. Angiogenesis, 2014; 17(4):921–933; doi: 10.1007/s10456-014-9439-0
23.
StaporPC, SweatRS, DashtiDC, et al.Pericyte dynamics during angiogenesis: New insights from new identities. J Vasc Res, 2014; 51(3):163–174; doi: 10.1159/000362276
NadineS, FernandesIJ, CorreiaCR, et al.Close-to-native bone repair via tissue-engineered endochondral ossification approaches. iScience, 2022; 25(11):105370; doi: 10.1016/j.isci.2022.105370
26.
ThompsonEM, MatsikoA, FarrellE, et al.Recapitulating endochondral ossification: A promising route to in vivo bone regeneration Emmet. J Tissue Eng Regen Med, 2015; 9(8):889–902; doi: 10.1002/term.1918
27.
GerstenfeldLC, CullinaneDM, BarnesGL, et al.Fracture healing as a post-natal developmental process: molecular, spatial, and temporal aspects of its regulation. J Cell Biochem, 2003; 884:873–884; doi: 10.1002/jcb.10435
28.
ZhangJ, PanJ, JingW. Motivating role of type H vessels in bone regeneration. Cell Prolif, 2020; 53(9):e12874; doi: 10.1111/cpr.12874
29.
YangL, TsangKY, TangHC, et al.Hypertrophic chondrocytes can become osteoblasts and osteocytes in endochondral bone formation. Proc Natl Acad Sci U S A, 2014; 111(33):12097–12102; doi: 10.1073/pnas.1302703111
30.
AghajanianP, MohanS. The art of building bone: Emerging role of chondrocyte-to-osteoblast transdifferentiation in endochondral ossification. Bone Res, 2018; 6(1):19; doi: 10.1038/s41413-018-0021-z
31.
BernhardJ, FergusonJ, RiederB, et al.Tissue-engineered hypertrophic chondrocyte grafts enhanced long bone repair. Biomaterials, 2017; 139:202–212; doi: 10.1016/j.biomaterials.2017.05.045
32.
NultyJ, BurdisR, KellyDJ. Biofabrication of prevascularised hypertrophic cartilage microtissues for bone tissue engineering. Front Bioeng Biotechnol, 2021; 9(June):661989–661916; doi: 10.3389/fbioe.2021.661989
33.
LinZ, ZhangX, FritchMR, et al.Engineering pre-vascularized bone-like tissue from human mesenchymal stem cells through simulating endochondral ossification. Biomaterials, 2022; 283(August 2021):121451; doi: 10.1016/j.biomaterials.2022.121451
34.
VailDJ, SomozaRA, CaplanAI. MicroRNA regulation of bone marrow mesenchymal stem cell chondrogenesis: Toward articular cartilage. Tissue Eng Part A, 2022; 28(5–6):254–269; doi: 10.1089/ten.tea.2021.0112
35.
LennonDP, CaplanAI. Isolation of rat marrow-derived mesenchymal stem cells. Exp Hematol, 2006; 34(11):1606–1607; doi: 10.1016/j.exphem.2006.07.015
36.
WuB, DurisinEK, DeckerJT, et al.Phosphate regulates chondrogenesis in a biphasic and maturation-dependent manner. Differentiation, 2017; 95(November 2016):54–62; doi: 10.1016/j.diff.2017.04.002
37.
SchottNG, VuH, StegemannJP. Multimodular vascularized bone construct comprised of vasculogenic and osteogenic microtissues. Biotechnol Bioeng, 2022; 119(11):3284–3296; doi: 10.1002/bit.28201
38.
de SilvaL, LongoniA, StaubliF, et al.Bone regeneration in a large animal model featuring a modular off-the-shelf soft callus mimetic. Adv Healthc Mater, 2023; 12(29):e2301717; doi: 10.1002/adhm.202301717
39.
MuellerM, TuanR. Functional characterization of hypertrophy in chondrogenesis. Arthritis Rheum, 2008; 58(5):1377–1388; doi: 10.1002/art.23370.Functional
40.
ScottiC, TonnarelliB, PapadimitropoulosA, et al.Recapitulation of endochondral bone formation using human adult mesenchymal stem cells as a paradigm for developmental engineering. Proc Natl Acad Sci U S A, 2010; 107(16):7251–7256; doi: 10.1073/pnas.1000302107
41.
KronenbergHM. Developmental regulation of the growth plate. Nature, 2003; 423(6937):332–336.
42.
AhmadAR, KaewpungsupP, KhorattanakulchaiN, et al.Recombinant human dentin matrix protein 1 (DMP1)induces the osteogenic differentiation of human periodontal ligament cells. Biotechnol Rep (Amst), 2019; 23:e00348; doi: 10.1016/j.btre.2019.e00348
43.
GerberHP, VuTH, RyanAM, et al.VEGF couples hypertrophic cartilage remodeling, ossification and angiogenesis during endochondral bone formation. Nat Med, 1999; 5(6):623–628; doi: 10.1038/9467
44.
BagnatoA, SpinellaF. Emerging role of endothelin-1 in tumor angiogenesis. Trends Endocrinol Metab, 2003; 14(1):44–50; doi: 10.1016/S1043-2760(02)00010-3
45.
HuDE, HileyCR, FanTPD. Comparative studies of the angiogenic activity of vasoactive intestinal peptide, endothelins-1 and -3 and angiotensin II in a rat sponge model. Br J Pharmacol, 1996; 117(3):545–551; doi: 10.1111/j.1476-5381.1996.tb15225.x
46.
NossinY, FarrellE, KoevoetWJLM, et al.Angiogenic potential of tissue engineered cartilage from human mesenchymal stem cells is modulated by indian hedgehog and Serpin E1. Front Bioeng Biotechnol, 2020; 8(April):327–313; doi: 10.3389/fbioe.2020.00327
47.
YoonYM, OhCD, KimDY, et al.Epidermal growth factor negatively regulates chondrogenesis of mesenchymal cells by modulating the protein kinase C-α, Erk-1, and p38 MAPK signaling pathways. J Biol Chem, 2000; 275(16):12353–12359; doi: 10.1074/jbc.275.16.12353
48.
KatoY, IwamotoM. Fibroblast growth factor is an inhibitor of chondrocyte terminal differentiation. J Biol Chem, 1990; 265(10):5903–5909; doi: 10.1016/s0021-9258(19)39448-7
49.
FreemanFE, AllenAB, StevensHY, et al.Effects of in vitro endochondral priming and pre-vascularisation of human MSC cellular aggregates in vivo. Stem Cell Res Ther, 2015; 6(218):218–218; doi: 10.1186/s13287-015-0210-2
