Systemic DKK1 neutralization enhances human adipose‐derived stem cell mediated bone repair

Abstract Progenitor cells from adipose tissue are able to induce bone repair; however, inconsistent or unreliable efficacy has been reported across preclinical and clinical studies. Soluble inhibitory factors, such as the secreted Wnt signaling antagonists Dickkopf‐1 (DKK1), are expressed to variable degrees in human adipose‐derived stem cells (ASCs), and may represent a targetable “molecular brake” on ASC mediated bone repair. Here, anti‐DKK1 neutralizing antibodies were observed to increase the osteogenic differentiation of human ASCs in vitro, accompanied by increased canonical Wnt signaling. Human ASCs were next engrafted into a femoral segmental bone defect in NOD‐Scid mice, with animals subsequently treated with systemic anti‐DKK1 or isotype control during the repair process. Human ASCs alone induced significant but modest bone repair. However, systemic anti‐DKK1 induced an increase in human ASC engraftment and survival, an increase in vascular ingrowth, and ultimately improved bone repair outcomes. In summary, anti‐DKK1 can be used as a method to augment cell‐mediated bone regeneration, and could be particularly valuable in the contexts of impaired bone healing such as osteoporotic bone repair.


| INTRODUCTION
Nonhealing skeletal defects are addressed in millions of surgeries worldwide each year, in diverse fields such as orthopedic, neurocranial, plastic, and oral and dental surgery. Segmental bone defects can be the result of congenital abnormalities, or can arise secondarily from diverse causes such as trauma, malignancy, or infection. In adult patients, a critical defect of long bones is defined as a bone loss involving >50% of the circumference or >2 cm in length. 1 The available techniques to surgical manage these conditions are mainly based on bone grafting, distraction osteogenesis, and the induced membrane technique. 2 These approaches require long times of recovery and Stefano Negri and Yiyun Wang contributed equally to this work. multiple surgeries. 3 The growing biomedical burden of skeletal defects coupled with the lack of adequate treatment options has fueled the interest in alternative therapies for bone regeneration, especially in the osteoporotic patient.
Adipose-derived stem cells (ASCs) have been used extensively to induce bone repair. 4,5 Although many experiences report a good potential of ASCs in bone healing, [6][7][8] batch-to-batch variability and cellular heterogeinity have been identified. 7,9,10 Indeed, contaminant nonprogenitor cells have been found to represent an obstacle to the osteogenic efficacy of ASC in several contexts. [11][12][13] Indeed, recent studies have observed that without additional biological augments, ASCs may have limited application in bone tissue engineering. 14,15 To circumvent this issue, stem/progenitor cell purification by cell sorting techniques has been published by our group and others. 9,16,17 However, the level of complexity for cell isolation is high, leading to regulatory challenges in clinical translation. A simpler solution would be to pharmacologically target those signaling pathways expressed in unpurified stromal cell population that may inhibit the process of osteogenic differentiation.
Dickkopf-1 (DKK1) is an extracellular Wnt antagonist regulating bone formation. Its impact on bone physiology is by competing with Wnt ligands for binding to coreceptors lipoprotein-related proteins 5 and 6 (LRP5 and LRP6). 18 Several studies have shown that DKK1 neutralizing antibodies (anti-DKK1) can accelerate bone formation and increase bone mineral density (BMD) in various animal models. 19 Systemic anti-DKK1 therapy has shown improved fracture healing capacity in rodent long bone fracture models. [20][21][22] The overall safety profile of anti-DKK1 has been confirmed in several preclinical models as well as in human clinical trials. [23][24][25] Recently, our laboratory reported that DKK1 is highly expressed in human ASCs, and anti-DKK1 improves the early osteogenic differentiation of human ASC in vitro. 26 Despite this accumulating translational evidence, the combination of anti-DKK1 with a stem/stromal cell therapy has not been examined in the context of in vivo bone repair.
In this study, anti-DKK1 treatment was examined as a means to improve outcomes associated with ASC mediated bone defect repair.
In order to assess this, human ASCs were engrafted into femoral segmental bone defect in NOD-Scid mice, with animals subsequently systemically treated with either anti-DKK1 or isotype control during the repair process. Overall, systemic anti-DKK1 induced an increase in human ASC engraftment and survival, an increase in vascular ingrowth, and ultimately improved bone repair outcomes.

| Osteogenic differentiation
Osteogenic differentiation medium consisted of DMEM, 10% FBS, 1% penicillin/streptomycin with 100 nM dexamethasone, 10 mM β-glycerophosphate, and 50 μM ascorbic acid (Sigma-Aldrich). Cells were cultured with osteogenic differentiation medium containing anti-DKK1 antibody or IgG isotype control. See Table S1 for antibody information. Medium was changed every 3 days. Alizarin red S (Sigma-Aldrich) staining was used to detect mineralization. Sodium hydroxide (0.1 N) was used to dissolve the calcium precipitate and quantified by absorbance at 548 nm. Mineralization on hydroxyapatite coated poly(lactic-co-glycolic acid) (HA-PLGA) scaffolds was evaluated in ASCs treated with IgG or anti-DKK1 in osteogenic medium for 24 hours before seeding. Next, the cellular scaffolds were cultured for 7 days and alizarin red staining was performed.

| RNA isolation and quantitative real-time polymerase chain reaction
TRIzol (Life Technology, Waltham, Massachusetts) was used for total RNA isolation. Then, according to the manufacturer's instructions,

Significance statement
Mesenchymal stem/stromal cell-mediated bone repair shows promise, yet inconsistent and incomplete tissue regeneration has been a persistent challenge. Here, systemic anti-DKK1 treatment improves upon progenitor cell mediated bone repair outcomes in a preclinical xenograft model.
In the future, DKK1 may represent a targetable molecular "brake" on the process of stem cell mediated bone formation, and release of this brake via neutralizing antibodies could be a method to improve bone repair outcomes. ANTI-DKK1 IMPROVES STEM CELL MEDIATED BONE REPAIR iScript cDNA Synthesis Kit (Bio-Rad, Hercules, California) was used to generate cDNA from RNA. SYBR Green PCR Master Mix (Life Technology) was used for quantitative real-time polymerase chain reaction (qRT-PCR). Primer information is provided in Table S2. N = 3 wells per group, and all studies were performed in three biological replicates.

| Scaffold preparation with ASC
Implants were prepared using 7.5 × 10 5 total ASC per scaffold. HA-PLGA scaffolds were custom fabricated using previously published methods 29  To evaluate the impact of DKK1 on cell adhesion, ASCs were pretreated either with anti-DKK1 or IgG in osteogenic medium for 24 hours. Then, 7.5 × 10 5 human ASC in 10 μL DMEM medium were seeded onto the scaffold and incubated at 37 C for 10 minutes. Each scaffold was placed into an individual well of a 48-well plate with 100 μL DMEM medium and incubated at 37 C for 6 hours. The unattached cells were then quantified.

| Animals and conditions
Twelve-week-old NOD-Scid male mice were used (strain code 001303, The Jackson Laboratories, Bar Harbor, Maine). Experimental procedures were consistent with ethical principles for animal research and were approved by Johns Hopkins University ACUC (protocol number MO18M144). Throughout the study, mice were housed in an IVC system rack using polypropylene cages (19 cm × 28 cm × 13 cm), with 12/12 night/day cycles, 21 C (±2 C) and 50% (±20%) relative humidity. All mice had ad libitum access to complete mouse food and filtered water. Animal allocation is described in Table S3. 2.6 | Surgical procedure A 3.5-mm mid-diaphyseal femoral segmental defect (FSD) was created and stabilized by plate osteosynthesis as previously described. 31 To perform the skeletal defect, animals were anesthetized with inhaled isoflurane (3%-5% induction, 2%-3% maintenance) delivered with combined oxygen and nitrous oxide (1:2 ratio) along with subdermal injection of sustained-release buprenorphine (1.2 mg/kg subcutaneous, q72h). Briefly, a 18 to 20 mm skin incision on the lateral aspect of the thigh. After the incision of the fascia lata, the interval between the vastus lateralis and biceps femoris muscles was identified and using a smooth periosteal elevator (Roboz Surgical Instrument Co., Maryland) the femoral diaphysis exposed.   Table S3. 2.8 | High-resolution roentgenography, dualenergy X-ray absorptiometry, and microcomputed tomography assessments Bone repair was assessed using a combination of high-resolution roentgenography (XR), dual-energy XR absorptiometry (DXA), and microcomputed tomography (μCT) imaging. First, the BMD of the defect site was prospectively analyzed every 4 weeks with DXA using a rectangular region of interest (ROI) centered within the femoral defect (Faxitron Bioptics, Tucson, Arizona). Serial quantification of BMD was also assessed within the lumbar spine, with a ROI encompassing the L1 to L6 vertebral bodies as well as the whole contralateral femur. Second, high-resolution XR imaging was also performed to survey bone healing every 4 weeks. Third, general morphological appearance and morphometric analysis were performed using ex vivo microCT using a Skyscan 1275 scanner (Bruker-MicroCT, Kontich, Belgium) with the following settings: 65 kV, 153 μA, 1 mm aluminum filter in 180 , six frames per 0.3 with a 9-μm voxel size. Images were reconstructed using NRecon. DataViewer software was used to realign the images and quantitative parameters were assessed using Skyscan CTan software (SkyScan, Kontich, Belgium) as previously published. 32 Briefly, a three-dimensional (3D) cylindrical ROI of 4.5 mm length and 2.5 mm diameter was set between the inner two screws of the plate. A threshold value range of 800 to 1250 Hounsfield units (HU) was used. 33,34 After global thresholding was carried out, a 3D data analysis, including bone volume (BV), bone volume/tissue volume (BV/TV), BMD, trabecular thickness (Tb.Th), and trabecular number (Tb.N), was performed.

| Quantification of osseous integration
The analysis of the bone-hardware interface was conducted following the guideline of Bruker for the quantification of bone around a metal implant (Method note MCT-074). Briefly, the plane of analysis was oriented orthogonally to the main axis of each screw. A volume of interest (VOI) mask was created based on the binary of the metal screw. This binary is filled and dilated to create an ROI surface at a constant distance from the metal surface, set by the pixel value of the dilation. In this manner, a hollow ring around the implant of 20 pixels thickness was obtained. After global thresholding, the BV of this ROI was measured using a 3D data analysis. All of the implanted screws of each animal were analyzed, and values reported as a mean value per animal.

| Statistical analysis
Results are expressed as the mean ± SD. Following an F test of the homogeneity of the variances, a Student's t test was used for twosample comparisons. A one-way analysis of variance (ANOVA) with Tukey's multiple comparisons test was used for more than two group comparisons (Graphpad Software 8.1). *P < .05 and **P < .01 were considered significant.

| Validation of cell seeding on a composite osteoinductive scaffold
Our approach was to improve upon ASC mediated regeneration of a long bone critical defect by the supplementation of anti-DKK1. In order to deliver human ASCs, we relied on a previously validated, osteoinductive, osteoconductive hydroxyapatite (H A)-coated poly(lactic-co-glycolic acid) (PLGA) composite scaffold. 16,29 Here, cylindrical HA-PLGA scaffolds were custom fabricated to fit a mouse femoral segmental defect (3.5 mm height, 2 mm diameter). ASC distribution, viability, and attachment onto scaffolds were next assessed ( Figure 1I-K). A homogenous distribution of cells throughout the scaffolds was observed, as shown by seeded scaffolds sectioned F I G U R E 1 Legend on next page. and stained with DAPI nuclear counterstaining ( Figure 1I). As shown by live-dead staining, high viability was observed at 1 hour after cell seeding ( Figure 1J), which was similar across treatment groups ( Figure S1). Cell attachment was observed by SEM, in which after 1 hour seeded ASCs had flattened and spread across the porous surface of the scaffold, with filopodial extensions observed ( Figure 1K).  Table S3 for a further summary of animal allocation, treatment regimens, and total cell numbers. *P < .05; **P < .01. ASCs, adipose-derived stem cells Anti-DKK1 did not significantly affect cell attachment, observed at 6 hours postseeding ( Figure S2). Contrarily, after 7 days of culture in osteogenic differentiation medium on HA-PLGA scaffolds, ASCs treated with anti-DKK1 showed increased mineral deposition (29.2% increase in comparison to IgG control; Figure S3).
Having further validated cell seeding protocols, we next sought to determine if anti-DKK1 treatment could improve upon ASC-mediated bone repair.  Table S3 for a further summary of animal allocation, treatment regimens, and total cell numbers. *P < .05; **P < .01. ASCs, adipose-derived stem cells treated defects with anti-DKK1 treatment showed a significant increase in BMD in comparison to other groups (66% increase in comparison to IgG control, 34% increase in comparison to anti-DKK1 alone, and 32% increase in comparison to ASC treated defects with IgG control). In comparison, anti-DKK1 did not increase the BMD at uninjured sites, assessed using both the contralateral femur and lumbar vertebrae (Table S4), a finding consistent with previously published reports in young mice. 35 In summary, systemic anti-DKK1

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improved ASC mediated FSD healing, as shown by increased BMD, and reduced size of the osteoectomy site.

| Anti-DKK1 promotes osteoblast differentiation and inhibits osteoclasts activity among FSDs
The osteoblast specific marker osteocalcin (OCN) was next evaluated across the different groups using immunofluorescent staining Osteoclast activity was next assessed using TRAP stained sections ( Figure S4). Representative images were again obtained from either the bone-scaffold interface ( Figure S4A-D) or central scaffold area ( Figure S4E-H). In general, ASC treated bone defects demonstrated significantly higher TRAP staining than that of acellular con-   Table S3 for a further summary of animal allocation, treatment regimens, and total cell numbers. White Scale bars = 50 μm. ASCs, adipose-derived stem cells. DAPI, 4 0 ,6-diamidino-2-phenylindole

| Anti-DKK1 increases ASC survival and enhances vascular ingrowth
Next, the potential effects of anti-DKK1 on ASC persistence within FSD sites were examined, using immunofluorescent staining for human-specific nuclei (HuNu) ( Figure 6). Use of acellular treatment groups without human cell implantations confirmed the specificity of staining ( Figure 6A). Systemic anti-DKK1 treatment led to a clear increase in the number of residual human cells at the study endpoint, which were found most frequently lining woven bone ( Figure 6B,C).

Quantification of HuNu immunostaining demonstrated a 92%
increase among anti-DKK1 treated animals ( Figure 6D) (*P < .05). Furthermore, co-immunohistochemical stainings for HuNu and OCN was performed ( Figure S5). These results showed a high degree of overlap between HuNu and OCN staining in the context of anti-DKK1 treatment.
One potential mechanism for increased ASC engraftment and were significantly upregulated after anti-DKK1 treatment ( Figure S6A,B, *P < .01) while MCL1 expression was not significantly affected ( Figure S6C).
Another potential mechanism for increased ASC engraftment and survival could be increased vascular ingrowth. To assess this, CD31 immunohistochemical staining was performed across defect sites

| DISCUSSION
Adipose-derived therapies have potential use for cell-augmented bone repair strategies. [4][5][6][7][8]39 Yet, inconsistent repair results in bone tissue engineering 40,41 have been linked to cell population heterogeneity, variability in cell preparation, or expression of osteogenic differentiation inhibitors. 11,13 Our observations suggest that DKK1 inhibits the early osteogenic differentiation of human ASCs, 26 44 and in instances of cartilage degeneration with arthritis. 38 It is likely that analogous findings were present in our stem cell xenograft model, in which systemic DKK1 led to a reduction in hASC apoptosis.
It is intriguing to speculate if this increase in stem cell survival is also related to vascular ingrowth. The majority of evidence shows that DKK1 inhibits in vitro and in vivo neoangiogenesis. 45,46 Reports indicate that this antiangiogenetic effect is by means of direct inhibition of endothelial cell proliferation and potentially by competitive binding of DKK2 to LRP6. 45 Here we found that anti-DKK1 conspicuously increased the vascularity among acellular groups, but did not obviously change vascular ingrowth between the two cell-treated groups.
More detailed studies must be performed in order to determine the relative contribution of increased vascular ingrowth to ASC survival within the early bone defect niche.
There are several limitations to the present study. First, to ensure consistent findings across animals, the in vivo experiment was performed with cells from a single female donor. Evidence suggests that ASC osteogenic differentiation potential differs on the basis of gender and location. 47 Further study must confirm the broader applicability of these results to human ASCs from donors with different demographics.
Second, our observations regarding the benefit of anti-DKK1 in bone repair were observed in young, adult male animals. It will be interesting to determine the extent to which this result should be true or even more prominent in animals with low bone mass or advanced age.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available on request from the corresponding author.