Cortical and Cancellous Bone Regeneration in Critical-sized Calvarial Defect

and Spinal Fusion Models Achieved Using Autologous Homologous Bone ConstructsPratima Labroo1, Nick Baetz2, Kendall Stauffer3, Michael Sieverts3, Jennifer Irvin1, Natalie Kirk3, Ian Robinson2, James Miess2,

Lyssa Lambert3, Caroline Garrett3, Anand Kumar4,5, Edward Swanson6, Nikolai Sopko1,2,3, Denver Lough1,2,3,6

Autologous bone grafts (ABG) remain the gold-standard forrepair of critical-sized cranial defects and lumbar spinal fusion.Healthy bone is transferred from a donor site, which can resultin donor site morbidity, hemorrhage, graft failure, and islimited by harvest amount, especially in pediatric applications.Products including demineralized bone matrix (DBM) andexogenous bone morphogenic protein-2 (BMP-2) have failedto regenerate complete cortical cancellous bone. Preclinicalevaluation of an autologous homologous bone construct(AHBC) comprised of living tissue has been developed toaddress these needs.

Introduction

1. Two - 8 mm diameter, full thickness, bilateral craniotomies were

created in 15 skeletally mature (6-7 month old) New Zealand White

(NZW) rabbits randomized for unilateral treatment (R or L

craniotomy) with split calvarial ABG, AHBC or DBM+BMP2 (10

ug/mL) + no treatment, open defect control (contralateral

craniotomy, all experimental groups).

2. Animals were imaged via CT immediately post-op and every 2 wks

thereafter through study termination at 8 wks post-op. Harvested

skulls underwent microCT imaging, gross imaging mechanical

testing (indentation), molecular analysis (Raman spectroscopy), and

scanning electron microscopy (SEM).

3. An additional 18 NZW rabbits were randomized to bilateral L4-L5

spinal fusion with ABG from the iliac crests, AHBC, or DBM+BMP2

(10ug/mL).

4. Animals were imaged via CT immediately post-op and every 2

weeks thereafter through study termination at 12 wks post-op when

vertebral columns were harvested.

5. Harvested lumbar spinal regions underwent physical manipulation

testing by two blinded raters to evaluate spinal fusion (0, no fusion;

1, partial fusion; 2, fused) followed by microCT imaging.

6. Spinal fusion masses underwent gross and microCT imaging,

compressive indentation test, Raman spectroscopy, and SEM.

7. Gene expression trends of processed samples was analyzed using

osteogenesis pathway PCR arrays.

Conclusions

1. AHBC treatment resulted in similar craniotomy closure and spinalfusion mass development compared to ABG with minimal andpoorly formed bone with DBM+BMP2 treatment on CT imagingand gross inspection.

2. Mechanical, compositional, and structural analysis demonstratedAHBC-formed bone was similar to ABG, whereas DBM+BMP2and untreated controls had properties more indicative of fibrosiswith minimal bone formation.

3. AHBC treatment is a novel therapy that allows for small harvestvolumes that can be obtained with minimally invasive techniques,which can generate functionally relevant cortical cancellous bonearchitecture in both craniotomy and spinal fusion models.

Methods

Figure 1: Heatmaps produced from hierarchical clustering of A. osteogenesis,B. wound healing, and C. angiogenesis pathway genes (y-axis) from 4 pre- and 5post-processed rabbit cranium samples (x-axis). Dark red and yellow areassociated with the highest and lowest levels of gene expression. Molecularcharacterization demonstrates that processing cranium into AHSC producesinherent changes in gene expression that may alter cellular function to upregulateregenerative markers and promote healing upon transfer back to patients.

Corresponding Author – PratimaLabroo@PolarityTE.com

1. Serial CT imaging showed increased closure of cranial defects and

development of spinal fusions with AHBC similar to ABG and both

were greater than DBM+BMP2, which had minimal fusion mass

development seen on CT, microCT, and gross inspection (Figure 2).

2. Cranial defects treated with ABG and AHBC had significantly higher

indentation modulus (6.4±5.8 and 6.3±10.1 MPa, respectively) and

indentation (ballistometer) (-0.05±0.02 and 0.09±0.02 mm,

respectively), compared to defects treated with DBM+BMP2 (3.5±2.4

MPa and 0.8±0.4 mm, respectively, p<0.05 vs DBM-BMP2) (Figure 3).

3. Gene expression analysis showed an upregulated of osteogenic

markers in AHBC treated specimen compared to autograft (Figure 5

and 10).

4. SEM imaging showed cortical and cancellous bone architecture in

AHBC treated craniotomies and spinal fusions qualitatively similar to

ABG, whereas DBM+BPM2 treatments resulted in scant and poor

bone architecture and fibrosis (Figure 6 and 11).

5. Physical manipulation demonstrated a fusion rate of 83.3% and a

mode value of 2 for both ABG and AHBC and a 0% fusion rate and

mode value of 0 for DBM+BMP2 (Figure 7).

6. Spinal masses from ABG and AHBC had significantly higher

indentation modulus (40.4±19.5 and 40.2±18.7 MPa, respectively)

and indentation (ballistometer) (-0.01±0.02 and 0.01±0.02 mm,

respectively) compared to masses treated with DBM+BMP2 (10.2±9.8

MPa and 0.5±0.4 mm, p<0.05 vs DBM+BMP2) (Figure 8).

7. Raman analysis of AHBC and autograft spinal treatments

demonstrated average spectra with a high intensity phosphate peak

at 961 cm-1 indicative of bone mineral hydroxyapatite formation while

DBM + BMP2 had low intensity 961 cm-1 peaks (Figure 9).

Results

Figure 8: Ex-vivo bone indentation modulus (stiffness) comparing autograft versusAHBC treated spinal fusion vs DBM+BMP2 treated spinal fusion. AHBC treated spinalfusion mass had comparable stiffness to Autograft treated spinal fusion mass.Indentation test was done using 1.5 mm diameter probe.

1 Department of Research & Development, Division of Biomedical Engineering, PolarityTE, Inc., Salt Lake City, Utah; 2 Department of Research & Development, Division of Cell and Tissue Biology, PolarityTE, Inc., Salt Lake City, Utah;

3 Department of Research & Development, Division of Translational Medicine and Product Development, PolarityTE, Inc., Salt Lake City, Utah; 4 UH Cleveland Medical Center, Department of Plastic and Reconstructive Surgery, Westlake, OH;

5 Clinical Advisory Board, PolarityTE, Inc, Salt Lake City, Utah; 6 Department of Clinical Operations, PolarityTE, Inc, Salt Lake City, Utah

Cranial Indentation Modulus

Figure 3: (Left) Ex-vivo bone indentation modulus (stiffness) comparing autograft versusAHBC treated cranial defect vs DBM treated defects. (Right) Load v. displacement graphshowing mechanical response of AHBC treated bone, autograft and DBM-treated bonecompared to native bone during indentation testing. Slope of the graph shows similarbone strength. Indentation test was done using 1.5 mm diameter probe.

Cranial AHBC Molecular Composition Analysis

Figure 4: Chemigram (chemical map with false coloring) showing hydroxyapatitedistribution across the treated cranial defect cross section determined using Ramanspectroscopy shows chemical composition of AHBC is similar to autograft and native bone.

Hydroxyapatite ChemigramReference Image

Figure 9: Raman spectra comparison of Autograft v. AHBC v. DBM+BMP2 treatedspinal fusion (labels show peak locations of interest such as hydroxyapatite andcollagen in the spectral fingerprint region).

Spinal Fusion MicroCT and Bone Mineral Density

Figure 2: MicroCT images of critically-sized cranial defects over 8-weeks. Autograft v.

AHBC v. DBM+BMP2 treatments. AHBC treatment shows formation of diploic architecture

across the cross-section. AHBC treatment had higher bone mineral density (BMD) and

trabecular BMD compared to DBM+BMP2 and untreated groups.

Cranial AHBC Molecular Profile (Autograft vs ex vivo AHBC)

Spinal Fusion Molecular Analysis

AutograftAHBCDMB+BMP2

Ram

an In

ten

sity

(CP

S)

Raman Shift (cm-1)

Figure 5: Heat maps displaying fold change differential gene expression analysis ofOsteogenesis pathway shows upregulation of wound healing and osteogenic factors inAHBC-generated bone compared to autograft.

Figure 7A: AHBC osteogenic regeneration observed in spinal defect.Red box = defect; Green box = AHBC-induced bone regeneration. Bone mineraldensity and fusion frequency shows AHBC response comparable to Autograft.Figure 7B: MicroCT images of critically-sized cranial defects post healing at 8-weeks. Autograft v. AHBC v. DBM+BMP2 treatments. Red boxes: Healing sites.

AHBC-Regenerated Bone

A. B. C. D. E.

Autograft

A. B. C. D. E.

DBM + BMP2

A. B. C. D. E.

Native Bone

A. B. C. D. E.

Autograft

AHBC

DBM +BMP2

Ind

en

tati

on

Mo

du

lus

(MP

a)

AHBC Molecular Profile (Native cranial bone vs ex vivo AHBC)

Native Defect POW 4 POW 7 POW 8

AHBC (Right)

Untreated (Left)

Autograft (Left)

Untreated (Right)

DBM + BMP2 (Left)

Untreated (Right)

Spinal Fusion Ultrastructure Imaging

Figure 11: Representative samples from each group imaged with Multiphoton, SEM,and DLSR. All samples are ex vivo POW12, and demonstrate that AHBC-generatedosseous spinal ultrastructure is analogous to autograft.

Spinal Fusion Ultrastructure Imaging

Autograft DBM + BMP2 AHBC

DSLR

SEM

Autograft DBM + BMP2 AHBC

MP

SEM

DSLR

Figure 10: Heat maps displaying fold change differential gene expression analysis ofosteogenesis pathway shows upregulation of osteogenic factors such as RUNX2 andBGLAP in AHBC-generated bone compared to autograft.

Cranial defect treatments – Microtomography

AHBC (Right)Untreated (Left)

Autograft (Left)Untreated (Right)

DBM + BMP2 (Left)Untreated (Right)

Figure 6: AHBC-regenerated bone tissue shows similar ultrastructure to nativebone tissues indicating full functional osseous neo-generation and cortico-cancellous cross-sections. A. Multiphoton, B. Confocal imaging (Green=LonzaOsteoimage stain, Red = actin red 555 stain, Blue = nuclear ‘NucBlue’ stain), C.Stereoscope, D. Scanning Electron (SEM) Backscatter detector, E. SEM C2DX.

Spinal Fusion Indentation Testing

Cranial defect Ultrastructure Imaging

A.

B.

Figure 12: Digital single lens reflex (DSLR) and scanning electron microscope (SEM)images of a representative set of fusion mass bodies after extraction from the spinalcolumn. Images show AHBC regenerates spinal bone ultrastructure similar to autograft.

Spinal Fusion Molecular profile (Autograft vs ex vivo AHBC)

Osteogenesis Wound healing Angiogenesis

C.B.A.

© 2019 PolarityTE, Inc. Patents Pending

Cranial Defect Rabbit Model

Spinal Defect Rabbit Model