Recapitulating bone development through engineered mesenchymal condensations and mechanical cues for tissue regeneration

Large bone defects cannot form a callus and exhibit high complication rates even with the best treatment strategies available. Tissue engineering approaches often use scaffolds designed to match the properties of mature bone. However, natural fracture healing is most efficient when it recapitulates development, forming bone via a cartilage intermediate (endochondral ossification). Because mechanical forces are critical for proper endochondral bone development and fracture repair, we hypothesized that recapitulating developmental mechanical forces would be essential for large bone defect regeneration in rats. Here, we engineered mesenchymal condensations that mimic the cellular organization and lineage progression of the early limb bud in response to local transforming growth factor–β1 presentation from incorporated gelatin microspheres. We then controlled mechanical loading in vivo by dynamically tuning fixator compliance. Mechanical loading enhanced mesenchymal condensation–induced endochondral bone formation in vivo, restoring functional bone properties when load initiation was delayed to week 4 after defect formation. Live cell transplantation produced zonal human cartilage and primary spongiosa mimetic of the native growth plate, whereas condensation devitalization before transplantation abrogated bone formation. Mechanical loading induced regeneration comparable to high-dose bone morphogenetic protein-2 delivery, but without heterotopic bone formation and with order-of-magnitude greater mechanosensitivity. In vitro, mechanical loading promoted chondrogenesis and up-regulated pericellular matrix deposition and angiogenic gene expression. In vivo, mechanical loading regulated cartilage formation and neovascular invasion, dependent on load timing. This study establishes mechanical cues as key regulators of endochondral bone defect regeneration and provides a paradigm for recapitulating developmental programs for tissue engineering.

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Recapitulating bone development for tissue regeneration through engineered mesenchymal condensations and mechanical cues

10.1101/157362 ◽

2017 ◽

Cited By ~ 2

Author(s):

Anna M. McDermott ◽

Samuel Herberg ◽

Devon E. Mason ◽

Hope B. Pearson ◽

James H. Dawahare ◽

...

Keyword(s):

Tissue Engineering ◽

Bone Formation ◽

Mechanical Loading ◽

Endochondral Ossification ◽

Bone Development ◽

Bone Defects ◽

Limb Bud ◽

Large Bone ◽

Large Bone Defects

ABSTRACTLarge bone defects cannot heal without intervention and have high complication rates even with the best treatments available. In contrast, bone fractures naturally healing with high success rates by recapitulating the process of bone development through endochondral ossification.1 Endochondral tissue engineering may represent a promising paradigm, but large bone defects are unable to naturally form a callus. We engineered mesenchymal condensations featuring local morphogen presentation (TGF-β1) to mimic the cellular organization and lineage progression of the early limb bud. As mechanical forces are 2,3 critical for proper endochondral ossification during bone morphogenesis2,3 and fracture healing, we hypothesized that mechanical cues would be important for endochondral regeneration.4,5 Here, using fixation plates that modulate ambulatory load transfer through dynamic tuning of axial compliance, we found that in vivo mechanical loading was necessary to restore bone function to large bone defects through endochondral ossification. Endochondral regeneration produced zonal cartilage and primary spongiosa mimetic of the native growth plate. Live human chondrocytes contributed to endochondral regeneration in vivo, while cell devitalization prior to condensation transplantation abrogated bone formation. Mechanical loading induced regeneration comparable to high-dose BMP-2 delivery, but without heterotopic bone formation and with order-of-magnitude greater mechanosensitivity.6–8In vitro, mechanical loading promoted chondrogenesis, and upregulated pericellular collagen 6 deposition and angiogenic gene expression. Consistently, in vivo mechanical loading regulated cartilage formation and neovascular invasion dependent on load timing. Together, this study represents the first demonstration of the effects of mechanical loading on transplanted cell-mediated bone defect regeneration, and provides a new template for recapitulating developmental programs for tissue engineering.

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Effects of in vivo mechanical loading on large bone defect regeneration

Journal of Orthopaedic Research® ◽

10.1002/jor.22042 ◽

2011 ◽

Vol 30 (7) ◽

pp. 1067-1075 ◽

Cited By ~ 72

Author(s):

Joel D. Boerckel ◽

Yash M. Kolambkar ◽

Hazel Y. Stevens ◽

Angela S.P. Lin ◽

Kenneth M. Dupont ◽

...

Keyword(s):

Mechanical Loading ◽

Bone Defect ◽

Large Bone Defect ◽

Large Bone

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Combinatorial morphogenetic and mechanical cues to mimic bone development for defect repair

10.1101/561837 ◽

2019 ◽

Author(s):

S. Herberg ◽

A. M. McDermott ◽

P. N. Dang ◽

D. S. Alt ◽

R. Tang ◽

...

Keyword(s):

Stem Cell ◽

Bone Formation ◽

Mechanical Loading ◽

Endochondral Ossification ◽

Bone Development ◽

Human Mesenchymal Stem Cell ◽

Bone Defects ◽

Mechanical Environment ◽

Tgf Β1

AbstractEndochondral ossification during long bone development and natural fracture healing initiates by mesenchymal cell condensation and is directed by local morphogen signals and mechanical cues. Here, we aimed to mimic these developmental conditions for regeneration of large bone defects. We hypothesized that engineered human mesenchymal stem cell (hMSC) condensations with in situ presentation of transforming growth factor-β1 (TGF-β1) and/or bone morphogenetic protein-2 (BMP-2) from encapsulated microparticles would promote endochondral regeneration of critical-sized rat femoral bone defects in a manner dependent on the in vivo mechanical environment. Mesenchymal condensations induced bone formation dependent on morphogen presentation, with dual BMP-2 + TGF-β1 fully restoring mechanical bone function by week 12. In vivo ambulatory mechanical loading, initiated at week 4 by delayed unlocking of compliant fixation plates, significantly enhanced the bone formation rate in the four weeks after load initiation in the dual morphogen group. In vitro, local presentation of either BMP-2 alone or BMP-2 + TGF-β1 initiated endochondral lineage commitment of mesenchymal condensations, inducing both chondrogenic and osteogenic gene expression through SMAD3 and SMAD5 signaling. In vivo, however, endochondral cartilage formation was evident only in the BMP-2 + TGF-β1 group and was enhanced by mechanical loading. The degree of bone formation was comparable to BMP-2 soaked on collagen but without the ectopic bone formation that limits the clinical efficacy of BMP-2/collagen. In contrast, mechanical loading had no effect on autograft-mediated repair. Together, this study demonstrates a biomimetic template for recapitulating developmental morphogenic and mechanical cues in vivo for tissue engineering.One Sentence SummaryMimicking aspects of the cellular, biochemical, and mechanical environment during early limb development, chondrogenically-primed human mesenchymal stem cell condensations promoted functional healing of critical-sized femoral defects via endochondral ossification, and healing rate and extent was a function of the in vivo mechanical environment.

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Decellularized bone extracellular matrix in skeletal tissue engineering

Biochemical Society Transactions ◽

10.1042/bst20190079 ◽

2020 ◽

Vol 48 (3) ◽

pp. 755-764

Author(s):

Benjamin B. Rothrauff ◽

Rocky S. Tuan

Keyword(s):

Tissue Engineering ◽

Bone Regeneration ◽

Tissue Regeneration ◽

Animal Studies ◽

Tumor Resection ◽

Regenerative Capacity ◽

Skeletal Tissue ◽

Large Bone

Bone possesses an intrinsic regenerative capacity, which can be compromised by aging, disease, trauma, and iatrogenesis (e.g. tumor resection, pharmacological). At present, autografts and allografts are the principal biological treatments available to replace large bone segments, but both entail several limitations that reduce wider use and consistent success. The use of decellularized extracellular matrices (ECM), often derived from xenogeneic sources, has been shown to favorably influence the immune response to injury and promote site-appropriate tissue regeneration. Decellularized bone ECM (dbECM), utilized in several forms — whole organ, particles, hydrogels — has shown promise in both in vitro and in vivo animal studies to promote osteogenic differentiation of stem/progenitor cells and enhance bone regeneration. However, dbECM has yet to be investigated in clinical studies, which are needed to determine the relative efficacy of this emerging biomaterial as compared with established treatments. This mini-review highlights the recent exploration of dbECM as a biomaterial for skeletal tissue engineering and considers modifications on its future use to more consistently promote bone regeneration.

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A novel gelatin/chitooligosaccharide/demineralized bone matrix composite scaffold and periosteum-derived mesenchymal stem cells for bone tissue engineering

Biomaterials Research ◽

10.1186/s40824-021-00220-y ◽

2021 ◽

Vol 25 (1) ◽

Author(s):

Thakoon Thitiset ◽

Siriporn Damrongsakkul ◽

Supansa Yodmuang ◽

Wilairat Leeanansaksiri ◽

Jirun Apinun ◽

...

Keyword(s):

Tissue Engineering ◽

Stem Cells ◽

Mesenchymal Stem Cells ◽

Bone Formation ◽

Bone Tissue Engineering ◽

Bone Tissue ◽

Demineralized Bone Matrix ◽

Bone Matrix ◽

Alp Activity

Abstract Background A novel biodegradable scaffold including gelatin (G), chitooligosaccharide (COS), and demineralized bone matrix (DBM) could play a significant part in bone tissue engineering. The present study aimed to investigate the biological characteristics of composite scaffolds in combination of G, COS, and DBM for in vitro cell culture and in vivo animal bioassays. Methods Three-dimensional scaffolds from the mixture of G, COS, and DBM were fabricated into 3 groups, namely, G, GC, and GCD using a lyophilization technique. The scaffolds were cultured with mesenchymal stem cells (MSCs) for 4 weeks to determine biological responses such as cell attachment and cell proliferation, alkaline phosphatase (ALP) activity, calcium deposition, cell morphology, and cell surface elemental composition. For the in vivo bioassay, G, GC, and GCD, acellular scaffolds were implanted subcutaneously in 8-week-old male Wistar rats for 4 weeks and 8 weeks. The explants were assessed for new bone formation using hematoxylin and eosin (H&E) staining and von Kossa staining. Results The MSCs could attach and proliferate on all three groups of scaffolds. Interestingly, the ALP activity of MSCs reached the greatest value on day 7 after cultured on the scaffolds, whereas the calcium assay displayed the highest level of calcium in MSCs on day 28. Furthermore, weight percentages of calcium and phosphorus on the surface of MSCs after cultivation on the GCD scaffolds increased when compared to those on other scaffolds. The scanning electron microscopy images showed that MSCs attached and proliferated on the scaffold surface thoroughly over the cultivation time. Mineral crystal aggregation was evident in GC and greatly in GCD scaffolds. H&E staining illustrated that G, GC, and GCD scaffolds displayed osteoid after 4 weeks of implantation and von Kossa staining confirmed the mineralization at 8 weeks in G, GC, and GCD scaffolds. Conclusion The MSCs cultured in GCD scaffolds revealed greater osteogenic differentiation than those cultured in G and GC scaffolds. Additionally, the G, GC, and GCD scaffolds could promote in vivo ectopic bone formation in rat model. The GCD scaffolds exhibited maximum osteoinductive capability compared with others and may be potentially used for bone regeneration.

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Bone defect reconstruction via endochondral ossification: A developmental engineering strategy

Journal of Tissue Engineering ◽

10.1177/20417314211004211 ◽

2021 ◽

Vol 12 ◽

pp. 204173142110042

Author(s):

Rao Fu ◽

Chuanqi Liu ◽

Yuxin Yan ◽

Qingfeng Li ◽

Ru-Lin Huang

Keyword(s):

Bone Regeneration ◽

Bone Defect ◽

Bone Repair ◽

Endochondral Ossification ◽

Current Knowledge ◽

Endochondral Bone ◽

Defect Reconstruction ◽

Hypoxic Conditions ◽

Bone Defect Reconstruction

Traditional bone tissue engineering (BTE) strategies induce direct bone-like matrix formation by mimicking the embryological process of intramembranous ossification. However, the clinical translation of these clinical strategies for bone repair is hampered by limited vascularization and poor bone regeneration after implantation in vivo. An alternative strategy for overcoming these drawbacks is engineering cartilaginous constructs by recapitulating the embryonic processes of endochondral ossification (ECO); these constructs have shown a unique ability to survive under hypoxic conditions as well as induce neovascularization and ossification. Such developmentally engineered constructs can act as transient biomimetic templates to facilitate bone regeneration in critical-sized defects. This review introduces the concept and mechanism of developmental BTE, explores the routes of endochondral bone graft engineering, highlights the current state of the art in large bone defect reconstruction via ECO-based strategies, and offers perspectives on the challenges and future directions of translating current knowledge from the bench to the bedside.

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Combination of optimized tissue engineering bone implantation with heel-strike like mechanical loading to repair segmental bone defect in New Zealand rabbits

Cell and Tissue Research ◽

10.1007/s00441-021-03458-z ◽

2021 ◽

Author(s):

Cong Zhu ◽

Jianbiao Lin ◽

Huixiang Jiang ◽

Jianting Gao ◽

Mingming Gao ◽

...

Keyword(s):

Tissue Engineering ◽

New Zealand ◽

Mechanical Loading ◽

Bone Defect ◽

Heel Strike ◽

Segmental Bone Defect ◽

Segmental Bone ◽

New Zealand Rabbits

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Conditional inactivation of VEGF-A in areas of collagen2a1 expression results in embryonic lethality in the heterozygous state

Development ◽

10.1242/dev.127.7.1445 ◽

2000 ◽

Vol 127 (7) ◽

pp. 1445-1453 ◽

Cited By ~ 5

Author(s):

J.J. Haigh ◽

H.P. Gerber ◽

N. Ferrara ◽

E.F. Wagner

Keyword(s):

Bone Formation ◽

Ischemic Cardiomyopathy ◽

Embryonic Lethality ◽

Limb Bud ◽

Endochondral Bone Formation ◽

Functional Importance ◽

Mouse Development ◽

Endochondral Bone ◽

Myocardial Layers ◽

Transgenic Lines

VEGF-A has been implicated in regulating the initial angiogenic invasion events that are essential for endochondral bone formation. VEGF-A mRNA expression was indeed found in the sclerotome of the developing somite and in the limb-bud mesenchyme at E10.5 in mouse development but declined during chondrogenesis and became upregulated in hypertrophic chondrocytes prior to angiogenic invasion. To determine the functional importance of VEGF-A expression in the developing chondrogenic tissues, VEGF-A was conditionally inactivated during early embryonic development using Collagen2a1-Cre transgenic lines. Deletion of a single VEGF-A allele in Collagen2a1-Cre-expressing cells results in embryonic lethality around E10.5. This lethality is characterized by aberrant development of the dorsal aorta and intersomitic blood vessels, along with defects in the developing endocardial and myocardial layers of the heart. A small percentage of VEGF(Flox)/+, Collagen2a1-Cre fetuses survive until E17.5, show aberrant endochondral bone formation and develop a heart phenotype resembling a dilated form of ischemic cardiomyopathy. These results provide insights into the function of VEGF-A in heart and endochondral bone formation and underscore the importance of tightly controlled levels of VEGF-A during development.

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