JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE RESEARCH J Tissue Eng Regen Med (2015) Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/term.2047
ARTICLE
Oxy133, a novel osteogenic agent, promotes bone regeneration in an intramembranous bone-healing model Andrew Li1†, Akishige Hokugo1†, Luis Andres Segovia1, Anisa Yalom1, Kameron Rezzadeh1, Situo Zhou1, Zheyu Zhang1, Farhad Parhami2, Frank Stappenbeck3 and Reza Jarrahy1* 1
Regenerative Bioengineering and Repair Laboratory, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA 3 MAX BioPharma Inc., Los Angeles, CA, USA 2
Abstract Current reconstructive techniques for complex craniofacial osseous defects are challenging and are associated with significant morbidity. Oxysterols are naturally occurring cholesterol oxidation products with osteogenic potential. In this study, we investigated the effects of a novel semi-synthetic oxysterol, Oxy133, on in vitro osteogenesis and an in vivo intramembranous bone-healing model. Rabbit bone marrow stromal cells (BMSCs) were treated with either Oxy133 or BMP-2. Alkaline phosphatase (ALP) activity, expression of osteogenic gene markers and in vitro mineralization were all examined. Next, collagen sponges carrying either Oxy133 or BMP-2 were used to reconstruct critical-sized cranial defects in mature rabbits and bone regeneration was assessed. To determine the mechanism of action of Oxy133 both in vitro and in vivo, rabbit BMSCs cultures and collagen sponge/Oxy133 implants were treated with the Hedgehog signalling pathway inhibitor, cyclopamine, and similar outcomes were measured. ALP activity in rabbit BMSCs treated with 1 μM Oxy133 was induced and was significantly higher than in control cells. These results were mitigated in cultures treated with cyclopamine. Expression of osteogenic gene markers and mineralization in BMSCs treated with 1 μM Oxy133 was significantly higher than in control groups. Complete bone regeneration was noted in vivo when cranial defects were treated with Oxy133; healing was incomplete, however, when cyclopamine was added. Collectively, these results demonstrate that Oxy133 has the ability to induce osteogenic differentiation in vitro in rabbit BMSCs and to promote robust bone regeneration in vivo in an animal model of intramembranous bone healing. Copyright © 2015 John Wiley & Sons, Ltd. Received 3 October 2014; Revised 10 April 2015; Accepted 29 April 2015
Keywords oxysterol; bone regeneration; rabbit; intramembranous bone healing; Hedgehog signalling; osteogenesis
1. Introduction Complex craniofacial defects that require bone reconstruction account for hundreds of millions of dollars in healthcare expenditures annually, and represent a therapeutic challenge to craniomaxillofacial surgeons. Autologous bone grafting – the traditional approach to the reconstruction of *Correspondence to: Reza Jarrahy, Division of Plastic and Reconstructive Surgery, Department of Surgery, David Geffen School of Medicine at UCLA, 200 UCLA Medical Plaza, Suite 465, Los Angeles, CA 90095-6960, USA. E-mail: [email protected] † These authors contributed equally to this study. Copyright © 2015 John Wiley & Sons, Ltd.
such defects – is associated with numerous shortcomings, including increased surgical costs, pain and morbidity associated with the bone donor site, graft infection and graft resorption. Moreover, available donor sites for autologous bone are inherently limited (Conway, 2010). Bone graft substitutes have been developed to address these limitations. Currently, the most clinically relevant of these capitalize on the potent osteoinductive properties of bone morphogenetic proteins (BMPs). However, the significant adverse side-effect profiles and exorbitant costs associated with BMPs limit their widespread use in clinical practice (Garrett et al., 2010; Shah et al., 2008). Despite this fact, few alternatives to BMPs have been developed for clinical use, neither
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has the potential of mesenchymal stem cells (MSCs) – which are an important focus of numerous regenerative medicine strategies – been adequately studied within the context of bone healing (Steinert et al., 2012). To respond to the gap in knowledge surrounding the roles of alternative osteoinductive growth factors and MSCs in bone regeneration, we have focused on the study of oxysterols and their effects on MSCs. Oxysterols are naturally occurring derivatives of cholesterol oxidation that possess osteogenic potential. We have previously observed that these non-toxic, biocompatible and relatively inexpensive molecules induce robust expression of markers of osteogenic differentiation in murine and human MSCs (Aghaloo et al., 2007; Kha et al., 2004). In addition, we have recently demonstrated that oxysterols induce osteogenic differentiation in vitro in rabbit MSCs (rBMSCs) as effectively as BMP-2 (Hokugo et al., 2013a, 2013b). These experiments were conducted using early analogues of synthetic oxysterols. Most recently, Montgomery et al. (2014) have described a novel semi-synthetic oxysterol, Oxy133, a structural analogue of the naturally occurring osteogenic oxysterol 20(S)-hydroxycholesterol, with more potent osteogenic activity and a simpler route of synthesis than any analogue previously studied. In this study we demonstrated the in vitro osteoinductive capacity of Oxy133 on rBMSCs and present, for the first time, evidence that this oxysterol can mediate successful healing of critical-sized calvarial defects in an in vivo model of intramembranous bone healing. We further explored the mechanisms by which Oxy133 exerts its in vitro and in vivo osteoinductive effects by studying the role of the Hedgehog (Hh) signalling pathway in these processes.
2. Materials and methods 2.1. Materials Ketamine hydrochloride (Ketaject®) was purchased from Phoenix Pharmaceutical Inc. (St. Joseph, MO, USA), xylazine (AnaSed®) was purchased from Lloyd Laboratories (Shenandoah, IW, USA), povidone–iodine antiseptic solution was purchased from Med-Vet International (Mettawa, IL, USA) and 1% lidocaine hydrochloric acid (HCl) with 1:100 000 epinephrine was obtained from Hospira Inc. (Lake Forest, IL, USA). α-Minimal essential
medium (α-MEM), L-glutamine and penicillin–streptomycin were purchased from Invitrogen (Carlsbad, CA, USA) and fetal bovine serum (FBS) was obtained from Omega Scientific Inc. (Tarzana, CA, USA). Recombinant human bone morphogenetic protein-2 (BMP-2) was obtained from eBioscience Inc. (San Diego, CA, USA). Total RNA isolation kit (RNeasy Plus Mini Kit) was obtained from Qiagen (Valencia, CA, USA) and reverse transcriptase and SYBR Green Supermix were purchased from Bio-Rad (Hercules, CA, USA). Ascorbic acid-2 phosphate, β-glycerolphosphate (BGP), dexamethasone (DEX), dimethyl sulphoxide (DMSO) and all other reagents were obtained from Sigma-Aldrich (St Louis, MO, USA). Collagen sponge was kindly supplied from Olympus Terumo Biomaterials Corp. (Tokyo, Japan). Oxy133 was provided by MAX BioPharma and synthesized as previously reported (Montgomery et al., 2014) (Figure 1).
2.2. Animals All animal procedures and protocols were reviewed and approved by the Animal Research Committee of the University of California, Los Angeles. New Zealand White rabbits were purchased from Charles River Laboratories (Wilmington, MA, USA) and were housed and maintained at the UCLA vivarium under the care of the veterinary staff, according to the regulations set forth by the UCLA Office of Protection of Research Subjects.
2.3. Isolation and culture of rabbit BMSCs Ten male New Zealand White rabbits (average weight 2.5 kg) were used for isolation of BMSCs. General anaesthesia was induced by intramuscular injection of ketamine hydrochloride (35 mg/kg) and xylazine (5 mg/kg). A 2 × 1 cm2 area along the iliac crest was shaved, disinfected with povidone– iodine antiseptic solution and injected with 1 ml 1% lidocaine HCl with 1:100 000 epinephrine. Using surface landmarks as a guide, approximately 1 ml marrow was aspirated from the ventral ilium, using an 18-gauge needle attached to a 5 ml syringe. The marrow aspirate was suspended in growth medium, consisting of α-MEM, 10% FBS, 2 mM L-glutamine and penicillin–streptomycin, and plated onto 10 cm culture dishes. Cells that were colonogenic and adherent were identified as the stromal cell fraction. These were detached and
Figure 1. Molecular structures of oxysterols 20(S), Oxy49 and Oxy133: 20S (left) represents the naturally occurring analogue; Oxy49 (centre) is synthesized by addition of an extra –OH group on C6, elimination of the double bond between C5 and C6 (lower circle on Oxy49) and addition of a double bond between C25 and C27 (upper circle on Oxy49); Oxy133 (right) is similar to Oxy49 but replaces the C25–C27 double bond with a one carbon side chain lengthening (upper circle on Oxy133) Copyright © 2015 John Wiley & Sons, Ltd.
J Tissue Eng Regen Med (2015) DOI: 10.1002/term
Oxy133 promotes bone regeneration
reseeded onto 15 cm culture dishes. The cells were allowed to reach 90% confluence before they were harvested or replated. The cells were expanded in medium containing 10% FBS, 2 mM L-glutamine and 1% penicillin–streptomycin. At passage 1 or 2, cells were seeded in multiwell plates and cultured with either: (a) control medium (CM) containing 5% FBS, 2 mM L-glutamine and 1% penicillin–streptomycin; (b) osteogenic medium (OM) containing CM with 3 mM BGP and 50 μg/ml ascorbic acid-2 phosphate; (c) treatment medium containing OM with 1 μM Oxy133; or (d) OM with 50 ng/ml rhBMP-2. The medi were changed every 72 h. All cell cultures were maintained at 37°C and 5% CO2.
2.4. Alkaline phosphatase (ALP) activity assay Following 4 days of cell culture under the various experimental conditions described above, we tested for ALP activity, an early marker of osteogenesis. Colorimetric ALP activity assays were performed as previously reported on whole cell extracts (Kha et al., 2004). Briefly, cells were rinsed twice with PBS and scraped into 200 μl lysis buffer (0.2% NP-40 in 1 M MgCl2) with a rubber policeman and sonicated for 5 s. Reaction solution containing alkaline buffer stock solution and phosphatase substrate was added to each well. This mixture was then incubated for 10–15 min at 37°C. The development of yellow colour was indicative of ALP activity. The reaction was stopped by the addition of 1 N sodium hydroxide (NaOH) to each well, and absorbance was determined at 405 nm. ALP activity was calculated using p-nitrophenol as a standard and normalized to total protein determined using the Bradford protein assay (Bradford, 1976).
2.5. Mineralization assays Following 21 days in culture under the various noted conditions, calcium deposits were stained with alizarin red. The cells were washed twice with PBS, fixed with 10% formalin in neutral buffer for 30 min, rinsed extensively with distilled water and then covered with alizarin red staining solution. The cells were incubated for 45 min and then rinsed with distilled water. The plates were finally rinsed extensively with PBS. Staining results were captured using a Nikon D40 digital camera (Nikon, Tokyo, Japan). Four independent cultures were evaluated in each of the groups. Moreover, matrix mineralization was quantified using the 45Ca incorporation assay, as previously reported (Kha et al., 2004).
2.6. Osteogenic gene expression After 4 days of cell culture, total RNA was extracted from cells using RNA isolation kits according to the manufacturers’ instructions. RNA was reverse-transcribed using reverse transcriptase to make single-stranded cDNA template; 1 μl template was mixed with iQ SYBR Green Supermix for quantitative real-time PCR assay, using a Copyright © 2015 John Wiley & Sons, Ltd.
Bio-Rad I-cycler IQ quantitative thermal cycler (Bio-Rad, Hercules, CA, USA). All PCR samples were prepared in duplicate wells of a 96-well plate. After 40 cycles of PCR, melting curves were examined to ensure primer specificity. Fold changes in gene expression were calculated using the –ΔΔCT method. Oligonucleotide primers were designed using Beacon Design 7 software. The primer sequences used were as follows: GAPDH, forward 5′-TGC ACC ACC AAC TGC TTA GC-3′, reverse 5′-GGC ATG GAC TGT GGT CAT GAG-3′, yielding a 87 bp product; Runt-related transcription factor 2 (RUNX2), forward 5′-GCC TTC AAG GTG GTA GCC C-3′, reverse 5′-CGT TAC CCG CCA TGA CAG TA-3′, yielding a 67 bp product; collagen type I (COL1), forward 5′-AGA GCA TGA CCG ATG GAT TC-3′, reverse 5′-CCT TCT TGA GGT TGC CAG TC3′, yielding a 177 bp product; and Osterix (OSX), forward 5′-CTC CAA GCG CTT CAC CCG GA-3′, reverse 5′-AGC TCC TTG GGA CCG GTG GCT-3′, yielding a 96 bp product.
2.7. In vivo intramembranous bone regeneration: rabbit calvarial bone defect model In vivo experiments were performed as previously reported (Hokugo et al., 2013a, 2013b). Male New Zealand White rabbits (average weight 4.0 kg) were used. A paramedian incision was made and the periosteum was reflected off the fronto-parietal skull. Bone defects of 8 mm in diameter (four defects/cranial bone) were carefully created with a trephine burr, and each of the four defects was randomly assigned to accommodate one of the following treatment options: collagen sponge incorporating 10 mg Oxy133 dissolved in ethanol; collagen sponge incorporating 1 mg Oxy133 dissolved in ethanol; collagen sponge incorporating ethanol only; collagen sponge incorporating 75 μg BMP-2; or no treatment. A minimum of six defects for each experimental condition was studied. The wounds were carefully closed with absorbable suture and a postoperative analgesic was administered for 3 days. Six weeks after implantation, the animals were sacrificed and the entire calvarium was harvested. The specimens were immediately fixed in 10% buffered formalin. The fixed tissue specimens were first examined by micro-computed tomography (micro-CT; mCT40, Scanco Medical, Bassersdorf, Switzerland) at an X-ray energy level of 55 kVp with a current of 72 mA. Threedimensional (3D) images of the cranial bones, including the areas of the reconstructed defects, were rendered and calculated, using a customized computational program, by digitally extracting the bone image using the predetermined threshold of 220. Micro-CT images were then scored for bone regeneration by three blinded and independent reviewers, using a previously described grading scale (Patel et al., 2008; Spicer et al., 2012) that accounts for the extent of bony projections as a measure of overall bone growth. Briefly, the scoring for extent of bony bridging and union was: score 0, no bone formation within defect; score 1, few bony spicules dispersed through defect; score 2, bony J Tissue Eng Regen Med (2015) DOI: 10.1002/term
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bridging only at defect borders; score 3, bony bridging over partial length of defect; and score 4, bony bridging within the entire span of the defect at its longest point.
2.8. Inhibition of Oxy133-mediated in vitro osteogenesis with the Hh signalling inhibitor cyclopamine The Hh signalling pathway has been shown to play a role in the process of osteogenesis in murine cells (Dwyer et al., 2007; Kim et al., 2007) and rabbit cells (Hokugo et al., 2013a, 2013b). In order to determine whether Hh signalling plays a role in the mechanism by which oxysterols modulate osteogenesis, cell cultures were treated with cyclopamine, a known inhibitor of the Hh pathway, prior to treatment with Oxy133. Rabbit BMSCs were pretreated for 2 h with control vehicle (DMSO) or cyclopamine at concentrations of 2 or 4 μM. Following pretreatment, 1 μM Oxy133 or control vehicle was added to cell cultures. After 4 days of incubation, ALP activity assays were performed as described above.
2.9. Inhibition of Oxy133-mediated in vivo bone regeneration with cyclopamine In order to determine whether Hh signalling plays a role in the mechanism by which Oxy133 modulates in vivo bone regeneration, we added 2 mg cyclopamine dissolved in ethanol to collagen sponges incorporating 10 mg (high dose) Oxy133 and implanted these into calvarial bone defects. Collagen sponges containing only 2 mg cyclopamine dissolved in ethanol were also implanted into separate defects to serve as controls. Six weeks after implantation, bone regeneration assessments were performed as described above.
2.10. Statistical analysis All in vitro experiments were repeated a minimum of three times. Statistical analyses were performed by oneway ANOVA with Dunnett’s multiple comparison of means tests; p < 0.05 was considered statistically significant.
3. Results 3.1. Effect of Oxy133 on rabbit BMSCs The osteogenic response of rBMSCs treated with osteogenic medium to Oxy133 was first evaluated by measurement of subsequent ALP activity levels. ALP activity was significantly elevated in rBMSCs exposed to Oxy133 and 50 ng/ml BMP-2 in comparison to control cells treated with vehicle alone. Oxy133 induced a greater response than 50 ng/ml BMP2 (Figure 2). Copyright © 2015 John Wiley & Sons, Ltd.
Figure 2. Rabbit BMSCs at confluence were treated with control medium (CM) or Oxy133 in osteogenic medium (OM). Positive control cultures were treated with 50 ng/ml BMP-2, known to activate ALP activity. After 4 days of incubation, ALP activity was determined in cell homogenates by a colorimetric assay. Data from a representative experiments are reported as the mean ± SD of quadruplicate determinations, normalized to protein concentration; *p < 0.05 vs CM
In order to further determine the effects of Oxy133 on osteogenic function in rBMSCs, we evaluated the ability of treated cells to form a mineralized matrix, using alizarin red staining. Cell cultures were maintained for 21 days and then assessed with alizarin red. Cells cultured in nonosteogenic medium (CM) demonstrated a negligible mineralization response, whereas areas of positive alizarin red staining were observed in cultures of cells treated with OM, Oxy133 and BMP-2. A significant increase in mineralization relative to OM cultures was noted in cells treated with Oxy133, as well as in cultures treated with BMP-2 (Figure 3A). In order to make a quantitative assessment of mineralization response, a 45Ca incorporation assay was performed. The amount of incorporated 45Ca was significantly higher in cultures treated with Oxy133 than that seen in controls and in cells treated with BMP2 (Figure 3B). In order to determine the effect of Oxy133 on the expression of genes of osteogenic differentiation in rBMSCs, the mRNA expression of key markers of osteogenesis, including RUNX2, COL and OSX, was examined. Following 4 days of treatment with OM supplemented with either Oxy133 or 50 ng/ml BMP-2, real-time PCR was performed. Treatment of rBMSCs with Oxy133 resulted in a significant increase in mRNA expression of all three gene markers relative to control, and this increase was either equivalent to or greater than the induction observed in cells treated with BMP-2 (Figure 4).
3.2. Bone regeneration in rabbit cranial bone defects To demonstrate the potential clinical relevance of these positive in vitro results, we used Oxy133 as a means of enhancing in vivo bone regeneration in an established J Tissue Eng Regen Med (2015) DOI: 10.1002/term
Oxy133 promotes bone regeneration
in animals treated with collagen sponge containing control vehicle only. By comparison, robust bone regeneration was observed in defects treated with collagen sponges incorporating either Oxy133 or BMP-2. Low-dose (1 mg) Oxy133 yielded substantial bone deposition, with some thin and immature bone centrally. However, complete bone healing was noted in specimens treated with high-dose (10 mg) Oxy133. These results were equivalent to those observed in defects treated with 75 μg BMP-2 (Figure 5A). When examined under higher magnification, mature lamellar bone (similar to naïve bone tissue) was observed within defects treated with collagen sponges incorporating either Oxy133 or BMP-2. It is important to note that there were no inflammatory cells in the vicinity of the regenerated bone (Figure 5B). Figure 5C shows the results of this scoring for regenerated bone at defects treated with various implants. Collagen sponges incorporating Oxy133 (10 mg) and collagen sponges incorporating BMP-2 received significantly higher scores for union compared to collagen sponge-only groups.
3.3. In vitro and in vivo inhibition of Oxy133mediated osteogenesis with cyclopamine
Figure 3. (A) Alizarin red staining for calcium mineralization in rBMSCs after 21 days of culture with control medium (CM), osteogenic medium (OM) and OM with 1 μM Oxy133 or 50 ng/ml BMP-2: representative images of alizarin red staining are shown; pink/red areas, background areas of calcium mineralization; black areas, dense mineral deposition. (B) Minerali45 zation was also quantified using a Ca incorporation assay; *p < 0.05 vs CM
animal model for craniofacial reconstruction. Histological and radiographic analyses of calvarial defect specimens harvested 6 weeks after implant placement revealed a persistent full-thickness defect in animals treated with no reconstruction. Some peripheral bone regrowth was noted
The role of the Hh signalling pathway in the osteoinductive activity of Oxy133 was assessed through Hh pathway inhibition in vitro and in vivo. Addition of 2 or 4 μM cyclopamine to rBMSC cell cultures consistently reversed the increases in ALP activity noted in cells treated with Oxy133; these doses were chosen based on unpublished data from our group, demonstrating the antagonistic role of cyclopamine in BMP-2-induced spinal fusion in rats. With cylcopamine treatment, ALP activity was decreased to a level similar to that seen in cells treated with OM only (Figure 6A). When cyclopamine was added to collagen sponges carrying 10 mg Oxy133 and implanted in calvarial defects, micro-CT and histology demonstrated significant attenuation of the bone regeneration originally noted in defects treated with Oxy133 (Figure 5). Rather than robust bone deposition and complete defect healing, new bone was
Figure 4. Oxy133 induces expression of markers of osteogenic differentiation in rBMSCs: BMSCs cultured to confluence in control medium (CM), osteogenic medium (OM), OM with 1 μM Oxy133 or OM with 50 ng/ml of BMP-2. After 4 days in culture, mRNA expression was measured by quantitative real-time PCR. Data from representative experiments are reported as mean ± SD of triplicate determinations; *p < 0.05 vs CM Copyright © 2015 John Wiley & Sons, Ltd.
J Tissue Eng Regen Med (2015) DOI: 10.1002/term
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Figure 5. (A) (Right) 3D renderings and (left) histological images (H&E staining) of rabbit cranial bone defects, 6 weeks after undergoing no reconstructive treatment (Defect) or after implantation with collagen sponge carrying control vehicle only (CS), collagen sponge incorporating 1 mg Oxy133 [CS + Oxy133 (1 mg)], collagen sponge incorporating 10 mg Oxy133 [CS + Oxy133 (10 mg)] or collagen sponge incorporating 75 μg BMP-2 (CS + BMP-2); bar = 1 mm. (B) Higher magnification of histological findings of the defect treated with collagen sponge incorporating 10 mg Oxy133 or BMP-2; bar = 100 μM. (C) Scoring for bony bridging and union within the defect treated with various implants; *p < 0.05 vs CS
only seen peripherally and central full-thickness defects persisted (Figure 6B). The results of the scoring for regenerated bone further supported this observation: defects Copyright © 2015 John Wiley & Sons, Ltd.
implanted with 10 mg Oxy133 plus cyclopamine received significantly lower scores than defects treated with 10 mg Oxy133 alone (Figure 6C). J Tissue Eng Regen Med (2015) DOI: 10.1002/term
Oxy133 promotes bone regeneration
Figure 6. (A) ALP activity is diminished by addition of cyclopamine (Cyc): rabbit BMSCs at confluence were pretreated with control vehicle (2 μM) or Cyc (4 μM) for 2 h; next, 1 μM Oxy133 was added to cell cultures; ALP activity was measured after 4 days; data are normalized to ALP activity in cells treated with CM; Tukey–Kramer multiple comparison test; *significant differences (p < 0.05) compared to Oxy133 1 μM. (B) (Left) 3D renderings and (right) histological images of rabbit cranial bone defects, 6 weeks after implantation with a collagen sponge incorporating 10 mg Oxy133 and 2 mg Cyc or collagen sponge incorporating 2 mg Cyc alone; bar = 1 mm. (C) Scoring for bony bridging and union within the defect treated with various implants; *p < 0.05 vs CS + 10 mg Oxy133
4. Discussion Among the growth factors that are effective in stimulating bone regeneration, BMPs are well-established and powerful osteoinductive agents (Urist, 1965). BMPs strongly induce osteogenic differentiation of mesenchymal stem cells and osteoblastic progenitor cells. Because of these qualities, BMPs have been central to the development of clinical strategies aimed at generating a viable bone graft substitute for use in the clinical setting. Commercial products incorporating recombinant human (rh)BMP-2 (InfuseTM, Medtronic, Minneapolis, MN, USA) and rhBMP7 (OP-1TM, Stryker Biotech, Hopkinton, MA, USA) have been approved for clinical use by the US Food and Drug Administration (FDA) in spine and maxillofacial surgery (Cahill et al., 2011). Numerous ’off-label’ clinical uses of BMP-2 have also been reported (Glied and Kraut, 2010; Copyright © 2015 John Wiley & Sons, Ltd.
Ong et al., 2010). However, the high concentrations of BMP used in these products have been associated with severe deleterious side-effects, including acute airway obstruction (Yaremchuk et al., 2010a, 2010b), heterotopic bone formation (Brower and Vickroy, 2008), sterile seroma (Garrett et al., 2010) and profound soft tissue oedema (Shah et al., 2008), prompting the FDA to issue a formal warning regarding these complications, which may be potentially life threatening. Moreover, these products are expensive and may cost more to implement in clinical therapies than traditional bone autografts (Buttermann, 2008). BMPs are further limited by their non-specific activity: they induce not only osteogenic but also adipogenic differentiation of mesenchymal stem cells by induction of PPARγ expression in vitro and in vivo (Kang et al., 2009). The quality of regenerated bone may therefore be suboptimal. Finally, rhBMPs are not approved for use in skeletally immature patients and therefore are not applicable to the treatment of paediatric patients, who account for a large number of the complex congenital craniofacial reconstructive cases that present to tertiary care referral centres. Considering the potential drawbacks associated with BMP-2 therapy, searching for other factors that can be used as alternatives to, or in conjunction with, BMP-2 to minimize the disadvantages associated with its use is a very worthwhile endeavour. To that end, we have studied oxysterols for their potential use in clinical bone regeneration therapies. Oxysterols comprise a large family of 27-carbon molecules that are oxygenated derivatives of cholesterol. They are naturally present in the vascular system and tissues of humans and other higher animals (Bjorkhem et al., 2002) and are involved in diverse biological processes, including cholesterol homeostasis, sphingolipid metabolism, platelet aggregation and apoptosis (Kha et al., 2004). We have found that oxysterols show promising bioactivity in bone tissue engineering (Amantea et al., 2008; Kha et al., 2004). We originally showed that a specific naturally occurring oxysterol, 20(S)-hydroxycholesterol (Figure 1, left), induces the osteogenic differentiation of murine mesenchymal stem cells while simultaneously inhibiting adipogenesis (Kha et al., 2004). From a series of structure– activity relationship studies, we further observed that the hydroxyl group on C20 of the side chain of 20(S)hydroxycholesterol is essential for activation of Hh signalling and induction of osteogenesis. We next described how Oxy49, a novel oxysterol analogue of 20 (S)-hydroxycholesterol (Figure 1, centre), induces osteogenesis in rBMSCs in vitro more robustly than previously described oxysterols (Hokugo et al., 2013a, 2013b). Most recently, we have synthesized another oxysterol analogue, Oxy133 (Figure 1, right), that has the highest osteoinductive potency observed to date, is more easily and inexpensively synthesized than other analogues and might therefore serve as a better candidate for large-scale development geared toward clinical translation (Montgomery et al., 2014). In this study we demonstrated the osteoinductive efficacy of Oxy133 both in vitro and in vivo. ALP activity J Tissue Eng Regen Med (2015) DOI: 10.1002/term
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was measured in cells that were cultured in CM and OM supplemented with various concentrations of Oxy133 and compared to cells treated witb BMP-2. The dose of BMP-2 used in this study was 50 ng/ml, which has previously been shown to induce ALP activity in rBMSCs (Roostaeian et al., 2006). ALP enzymatic activity is indicative of successful synthesis of a functional ALP protein, which suggests differentiation along an osteogenic pathway (Mornet et al., 2001; Siffert, 1951). The induction of ALP activity in rBMSCs by 1 μM Oxy133 was greater than the activity induced by BMP-2. This observation demonstrates the potent osteoinductive activity of Oxy133 in this in vitro rBMSC system and is consistent with similar results found in murine MSC cultures treated with oxysterol (Kha et al., 2004; Mackie, 2003). We also included a comparison of the effects of Oxy133 to Oxy49 to evaluate its osteoinductive activity relative to that of the earlier generation oxysterol analogue, which we have previously shown has potent in vitro and in vivo osteogenic effects (Montgomery et al., 2014). Oxy133 induced a significantly greater amount of ALP activity compared to Oxy49, confirming its greater osteognenic potential. Mineral deposition begins during extracellular matrix maturation, and is an indicator of the late stages of osteogenic differentiation (Kim et al., 2007; Mornet et al., 2001). Mineralization assays, therefore, not only provide confirmation of cellular commitment to an osteoblastic differentiation pathway but also provide an assessment of the functionality of the mature cell (Lizard et al., 1996). In this study, cells treated with 1 μM Oxy133 showed a significantly increased capacity for mineralization compared to controls, and compared to cells treated with BMP-2. We also examined the effect of Oxy133 on the expression of genes associated with osteogenic differentiation. RUNX2 expression is indicative of the early commitment of MSCs to osteochondrogenic progenitors, whereas terminal differentiation is linked to the expression of OSX (Satija et al., 2007). The majority of the organic matrix of bone is made up of COL1 (Lee et al., 2007), the expression of which has previously been found to occur at high levels in the early stages of osteogenesis (Denhardt et al., 2001). We have demonstrated that 1 μM Oxy133 consistently and significantly increases mRNA expression of all these markers in rBMSCs. These results, along with our ALP activity data, collectively suggest that, at an ideal concentration of 1 μM, Oxy133 is able to induce in vitro differentiation of rBMSCs along an osteogenic pathway. This study used 8 mm calvarial defects to evaluate bone regeneration in skeletally mature rabbits. Although the 8 mm defect used in this study is smaller than the 15 mm rabbit cranial defect favoured by some authors (Hollinger and Kleinschmidt, 1990), healing was very limited in the absence of any treatment, indicating that the 8 mm defect size was incapable of complete spontaneous healing at week 6. Previous studies have also reported incomplete spontaneous healing in 5 mm (Hokugo et al., 2005; Meikle et al., 1994) and 6 mm (Hokugo et al., 2013a, Copyright © 2015 John Wiley & Sons, Ltd.
2013b) calvarial defects in rabbits, suggesting that an 8 mm cranial bone defect is an appropriate model to assess the regenerative capacity of an osteoinductive agent. In our model, we did not observe radiographic or histological evidence of complete bone regeneration in defects that were left untreated. Defects treated with collagen sponges incorporating a control vehicle demonstrated very limited amounts of peripheral bone formation on micro-CT compared to the defects with sponges containing Oxy133 or BMP-2. By comparison, bone regeneration was clearly observed in defects implanted with sponges containing Oxy133 or BMP-2. Low-dose Oxy133 yielded incomplete bone healing, while the higher dose resulted in complete healing of the defect. The bones of the craniofacial skeleton develop via intramembranous ossification through condensation of mesenchymal tissue into vascularized connective tissue and subsequent mineralization (Sadler and Langman, 2012). This process is mediated by various biochemical signals, including parathyroid hormone-related protein (PTHrP), vascular endothelial growth factor (VEGF), matrix metalloproteinase 13 (MMP13), Noggin and BMP-2 (Mackie et al., 2011). We have previously observed the induction of robust osteogenic responses by Oxy133 in murine embryonic fibroblasts and marrow stromal cells, as well as in primary human mesenchymal stem cells (Montgomery et al., 2014). Given these findings, it is possible that in our in vivo defect model, both undifferentiated mesenchymal stem cells that accumulate at the site of injury and resident calvarial preosteoblasts within the adjacent periosteum, are induced to differentiate along osteogenic pathways under the influence of Oxy133, perhaps mimicking the role of BMP-2 in the process of intramembranous ossification. An understanding of the mechanisms by which this process unfolds is integral to understanding it more fully and developing its translational potential. Hh signalling has been noted to play a role in the healing of cranial bone. Specifically, Indian Hh is the critical factor in intramembranous calvarial ossification (Lenton et al., 2011), while Sonic Hh is a well known potent osteogenic factor in various cell types and experimental model systems (Abzhanov et al., 2007). The activation of the Hh signalling pathway by oxysterols potentially influences intramembranous bone formation, and this warrants evaluation. Our in vitro results show that when oxysteroltreated rBMSCs are exposed to cyclopamine, a known inhibitor of the Hh signalling pathway, ALP activity is significantly decreased (Figure 6A). We have observed this phenomenon with two distinct analogues of oxysterol, Oxy133 and Oxy49. Our findings indicate that Hh signalling plays a role in Oxy133-mediated osteogenesis in rBMSCs. In vivo, we noted significant attenuation of Oxy133-induced bone healing in calvarial defects treated with both Oxy133 and cyclopamine, again suggesting that Hh not only plays a role in the in vitro processes that drive osteogenesis, but that its role also has potential clinical significance (Figure 6B, C). Our studies clearly demonstrate that Oxy133, and in turn activation of Hh J Tissue Eng Regen Med (2015) DOI: 10.1002/term
Oxy133 promotes bone regeneration
signalling, appear to induce osteogenesis in the context of both intramembrenous bone formation (calvarial bone regeneration) and endochondral bone formation (spinal fusion) (Montgomery et al., 2014) that we have previously reported. Further study is needed to fully elucidate the relationship between oxysterols and Hh in bone regeneration.
5. Conclusions Oxysterols represent a readily available, naturally occurring, inert and relatively inexpensive material upon which a tissue-engineering solution to an elusive bone graft substitute can potentially be based. Semi-synthetic oxysterol analogues have been shown to support osteogenesis. We have demonstrated that Oxy133, a novel analogue of naturally occurring oxysterols, has potent osteoinductive capacity in both in vitro and in vivo models. Moreover, we have initial insights into the mechanism by which it exerts its osteogenic effects. Oxysterols have the potential to help us to address complex craniofacial defects in a manner that minimizes costs and morbidity without compromising patient outcomes. Further investigation
into the translational applicability of this class of compounds is currently under way.
Conflict of interest As the Scientific Founder of MAX BioPharma, Dr Parhami has a financial interest in Oxy133, which has been licensed by the company from UCLA for further development. Dr Stappenbeck is Director of Chemistry at MAX BioPharma and therefore has financial interests in the technology used in this study.
Acknowledgements The authors are especially grateful for contributions to animal experiments from Dr Wei Zhou, Department of Anesthesiology, David Geffen School of Medicine at UCLA. This study was supported by grants from the Annenberg Foundation for Craniofacial Surgery and Research at the University of California Los Angeles, Komedyplast Craniofacial Research, the Cleft Plate Foundation/Craniofacial Anomalies Research and the PSF National Endowment for Plastic Surgery.
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rat critical size calvarial defect. Nat Protoc 7: 1918–1929. Steinert AF, Rackwitz L, Gilbert F et al. 2012; Concise review: the clinical application of mesenchymal stem cells for musculoskeletal regeneration: current status and perspectives. Stem Cells Transl Med 1: 237–247. Urist MR 1965; Bone: formation by autoinduction. Science 150: 893–899. Yaremchuk KL, Toma MS, Somers ML, et al. 2010a; Acute airway obstruction in cervical spinal procedures with bone morphogenetic proteins. Laryngoscope 120: 1954–1957. Yaremchuk K, Toma M, Somers M 2010b; Acute airway obstruction associated with the use of bone-morphogenetic protein in cervical spinal fusion. Laryngoscope 120(suppl 4): S140.
Supporting information Additional supporting information may be found in the online version of this article at the publisher’s web-site.
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J Tissue Eng Regen Med (2015) DOI: 10.1002/term
ARTICLE
Oxy133, a novel osteogenic agent, promotes bone regeneration in an intramembranous bone-healing model Andrew Li1†, Akishige Hokugo1†, Luis Andres Segovia1, Anisa Yalom1, Kameron Rezzadeh1, Situo Zhou1, Zheyu Zhang1, Farhad Parhami2, Frank Stappenbeck3 and Reza Jarrahy1* 1
Regenerative Bioengineering and Repair Laboratory, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA 3 MAX BioPharma Inc., Los Angeles, CA, USA 2
Abstract Current reconstructive techniques for complex craniofacial osseous defects are challenging and are associated with significant morbidity. Oxysterols are naturally occurring cholesterol oxidation products with osteogenic potential. In this study, we investigated the effects of a novel semi-synthetic oxysterol, Oxy133, on in vitro osteogenesis and an in vivo intramembranous bone-healing model. Rabbit bone marrow stromal cells (BMSCs) were treated with either Oxy133 or BMP-2. Alkaline phosphatase (ALP) activity, expression of osteogenic gene markers and in vitro mineralization were all examined. Next, collagen sponges carrying either Oxy133 or BMP-2 were used to reconstruct critical-sized cranial defects in mature rabbits and bone regeneration was assessed. To determine the mechanism of action of Oxy133 both in vitro and in vivo, rabbit BMSCs cultures and collagen sponge/Oxy133 implants were treated with the Hedgehog signalling pathway inhibitor, cyclopamine, and similar outcomes were measured. ALP activity in rabbit BMSCs treated with 1 μM Oxy133 was induced and was significantly higher than in control cells. These results were mitigated in cultures treated with cyclopamine. Expression of osteogenic gene markers and mineralization in BMSCs treated with 1 μM Oxy133 was significantly higher than in control groups. Complete bone regeneration was noted in vivo when cranial defects were treated with Oxy133; healing was incomplete, however, when cyclopamine was added. Collectively, these results demonstrate that Oxy133 has the ability to induce osteogenic differentiation in vitro in rabbit BMSCs and to promote robust bone regeneration in vivo in an animal model of intramembranous bone healing. Copyright © 2015 John Wiley & Sons, Ltd. Received 3 October 2014; Revised 10 April 2015; Accepted 29 April 2015
Keywords oxysterol; bone regeneration; rabbit; intramembranous bone healing; Hedgehog signalling; osteogenesis
1. Introduction Complex craniofacial defects that require bone reconstruction account for hundreds of millions of dollars in healthcare expenditures annually, and represent a therapeutic challenge to craniomaxillofacial surgeons. Autologous bone grafting – the traditional approach to the reconstruction of *Correspondence to: Reza Jarrahy, Division of Plastic and Reconstructive Surgery, Department of Surgery, David Geffen School of Medicine at UCLA, 200 UCLA Medical Plaza, Suite 465, Los Angeles, CA 90095-6960, USA. E-mail: [email protected] † These authors contributed equally to this study. Copyright © 2015 John Wiley & Sons, Ltd.
such defects – is associated with numerous shortcomings, including increased surgical costs, pain and morbidity associated with the bone donor site, graft infection and graft resorption. Moreover, available donor sites for autologous bone are inherently limited (Conway, 2010). Bone graft substitutes have been developed to address these limitations. Currently, the most clinically relevant of these capitalize on the potent osteoinductive properties of bone morphogenetic proteins (BMPs). However, the significant adverse side-effect profiles and exorbitant costs associated with BMPs limit their widespread use in clinical practice (Garrett et al., 2010; Shah et al., 2008). Despite this fact, few alternatives to BMPs have been developed for clinical use, neither
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has the potential of mesenchymal stem cells (MSCs) – which are an important focus of numerous regenerative medicine strategies – been adequately studied within the context of bone healing (Steinert et al., 2012). To respond to the gap in knowledge surrounding the roles of alternative osteoinductive growth factors and MSCs in bone regeneration, we have focused on the study of oxysterols and their effects on MSCs. Oxysterols are naturally occurring derivatives of cholesterol oxidation that possess osteogenic potential. We have previously observed that these non-toxic, biocompatible and relatively inexpensive molecules induce robust expression of markers of osteogenic differentiation in murine and human MSCs (Aghaloo et al., 2007; Kha et al., 2004). In addition, we have recently demonstrated that oxysterols induce osteogenic differentiation in vitro in rabbit MSCs (rBMSCs) as effectively as BMP-2 (Hokugo et al., 2013a, 2013b). These experiments were conducted using early analogues of synthetic oxysterols. Most recently, Montgomery et al. (2014) have described a novel semi-synthetic oxysterol, Oxy133, a structural analogue of the naturally occurring osteogenic oxysterol 20(S)-hydroxycholesterol, with more potent osteogenic activity and a simpler route of synthesis than any analogue previously studied. In this study we demonstrated the in vitro osteoinductive capacity of Oxy133 on rBMSCs and present, for the first time, evidence that this oxysterol can mediate successful healing of critical-sized calvarial defects in an in vivo model of intramembranous bone healing. We further explored the mechanisms by which Oxy133 exerts its in vitro and in vivo osteoinductive effects by studying the role of the Hedgehog (Hh) signalling pathway in these processes.
2. Materials and methods 2.1. Materials Ketamine hydrochloride (Ketaject®) was purchased from Phoenix Pharmaceutical Inc. (St. Joseph, MO, USA), xylazine (AnaSed®) was purchased from Lloyd Laboratories (Shenandoah, IW, USA), povidone–iodine antiseptic solution was purchased from Med-Vet International (Mettawa, IL, USA) and 1% lidocaine hydrochloric acid (HCl) with 1:100 000 epinephrine was obtained from Hospira Inc. (Lake Forest, IL, USA). α-Minimal essential
medium (α-MEM), L-glutamine and penicillin–streptomycin were purchased from Invitrogen (Carlsbad, CA, USA) and fetal bovine serum (FBS) was obtained from Omega Scientific Inc. (Tarzana, CA, USA). Recombinant human bone morphogenetic protein-2 (BMP-2) was obtained from eBioscience Inc. (San Diego, CA, USA). Total RNA isolation kit (RNeasy Plus Mini Kit) was obtained from Qiagen (Valencia, CA, USA) and reverse transcriptase and SYBR Green Supermix were purchased from Bio-Rad (Hercules, CA, USA). Ascorbic acid-2 phosphate, β-glycerolphosphate (BGP), dexamethasone (DEX), dimethyl sulphoxide (DMSO) and all other reagents were obtained from Sigma-Aldrich (St Louis, MO, USA). Collagen sponge was kindly supplied from Olympus Terumo Biomaterials Corp. (Tokyo, Japan). Oxy133 was provided by MAX BioPharma and synthesized as previously reported (Montgomery et al., 2014) (Figure 1).
2.2. Animals All animal procedures and protocols were reviewed and approved by the Animal Research Committee of the University of California, Los Angeles. New Zealand White rabbits were purchased from Charles River Laboratories (Wilmington, MA, USA) and were housed and maintained at the UCLA vivarium under the care of the veterinary staff, according to the regulations set forth by the UCLA Office of Protection of Research Subjects.
2.3. Isolation and culture of rabbit BMSCs Ten male New Zealand White rabbits (average weight 2.5 kg) were used for isolation of BMSCs. General anaesthesia was induced by intramuscular injection of ketamine hydrochloride (35 mg/kg) and xylazine (5 mg/kg). A 2 × 1 cm2 area along the iliac crest was shaved, disinfected with povidone– iodine antiseptic solution and injected with 1 ml 1% lidocaine HCl with 1:100 000 epinephrine. Using surface landmarks as a guide, approximately 1 ml marrow was aspirated from the ventral ilium, using an 18-gauge needle attached to a 5 ml syringe. The marrow aspirate was suspended in growth medium, consisting of α-MEM, 10% FBS, 2 mM L-glutamine and penicillin–streptomycin, and plated onto 10 cm culture dishes. Cells that were colonogenic and adherent were identified as the stromal cell fraction. These were detached and
Figure 1. Molecular structures of oxysterols 20(S), Oxy49 and Oxy133: 20S (left) represents the naturally occurring analogue; Oxy49 (centre) is synthesized by addition of an extra –OH group on C6, elimination of the double bond between C5 and C6 (lower circle on Oxy49) and addition of a double bond between C25 and C27 (upper circle on Oxy49); Oxy133 (right) is similar to Oxy49 but replaces the C25–C27 double bond with a one carbon side chain lengthening (upper circle on Oxy133) Copyright © 2015 John Wiley & Sons, Ltd.
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reseeded onto 15 cm culture dishes. The cells were allowed to reach 90% confluence before they were harvested or replated. The cells were expanded in medium containing 10% FBS, 2 mM L-glutamine and 1% penicillin–streptomycin. At passage 1 or 2, cells were seeded in multiwell plates and cultured with either: (a) control medium (CM) containing 5% FBS, 2 mM L-glutamine and 1% penicillin–streptomycin; (b) osteogenic medium (OM) containing CM with 3 mM BGP and 50 μg/ml ascorbic acid-2 phosphate; (c) treatment medium containing OM with 1 μM Oxy133; or (d) OM with 50 ng/ml rhBMP-2. The medi were changed every 72 h. All cell cultures were maintained at 37°C and 5% CO2.
2.4. Alkaline phosphatase (ALP) activity assay Following 4 days of cell culture under the various experimental conditions described above, we tested for ALP activity, an early marker of osteogenesis. Colorimetric ALP activity assays were performed as previously reported on whole cell extracts (Kha et al., 2004). Briefly, cells were rinsed twice with PBS and scraped into 200 μl lysis buffer (0.2% NP-40 in 1 M MgCl2) with a rubber policeman and sonicated for 5 s. Reaction solution containing alkaline buffer stock solution and phosphatase substrate was added to each well. This mixture was then incubated for 10–15 min at 37°C. The development of yellow colour was indicative of ALP activity. The reaction was stopped by the addition of 1 N sodium hydroxide (NaOH) to each well, and absorbance was determined at 405 nm. ALP activity was calculated using p-nitrophenol as a standard and normalized to total protein determined using the Bradford protein assay (Bradford, 1976).
2.5. Mineralization assays Following 21 days in culture under the various noted conditions, calcium deposits were stained with alizarin red. The cells were washed twice with PBS, fixed with 10% formalin in neutral buffer for 30 min, rinsed extensively with distilled water and then covered with alizarin red staining solution. The cells were incubated for 45 min and then rinsed with distilled water. The plates were finally rinsed extensively with PBS. Staining results were captured using a Nikon D40 digital camera (Nikon, Tokyo, Japan). Four independent cultures were evaluated in each of the groups. Moreover, matrix mineralization was quantified using the 45Ca incorporation assay, as previously reported (Kha et al., 2004).
2.6. Osteogenic gene expression After 4 days of cell culture, total RNA was extracted from cells using RNA isolation kits according to the manufacturers’ instructions. RNA was reverse-transcribed using reverse transcriptase to make single-stranded cDNA template; 1 μl template was mixed with iQ SYBR Green Supermix for quantitative real-time PCR assay, using a Copyright © 2015 John Wiley & Sons, Ltd.
Bio-Rad I-cycler IQ quantitative thermal cycler (Bio-Rad, Hercules, CA, USA). All PCR samples were prepared in duplicate wells of a 96-well plate. After 40 cycles of PCR, melting curves were examined to ensure primer specificity. Fold changes in gene expression were calculated using the –ΔΔCT method. Oligonucleotide primers were designed using Beacon Design 7 software. The primer sequences used were as follows: GAPDH, forward 5′-TGC ACC ACC AAC TGC TTA GC-3′, reverse 5′-GGC ATG GAC TGT GGT CAT GAG-3′, yielding a 87 bp product; Runt-related transcription factor 2 (RUNX2), forward 5′-GCC TTC AAG GTG GTA GCC C-3′, reverse 5′-CGT TAC CCG CCA TGA CAG TA-3′, yielding a 67 bp product; collagen type I (COL1), forward 5′-AGA GCA TGA CCG ATG GAT TC-3′, reverse 5′-CCT TCT TGA GGT TGC CAG TC3′, yielding a 177 bp product; and Osterix (OSX), forward 5′-CTC CAA GCG CTT CAC CCG GA-3′, reverse 5′-AGC TCC TTG GGA CCG GTG GCT-3′, yielding a 96 bp product.
2.7. In vivo intramembranous bone regeneration: rabbit calvarial bone defect model In vivo experiments were performed as previously reported (Hokugo et al., 2013a, 2013b). Male New Zealand White rabbits (average weight 4.0 kg) were used. A paramedian incision was made and the periosteum was reflected off the fronto-parietal skull. Bone defects of 8 mm in diameter (four defects/cranial bone) were carefully created with a trephine burr, and each of the four defects was randomly assigned to accommodate one of the following treatment options: collagen sponge incorporating 10 mg Oxy133 dissolved in ethanol; collagen sponge incorporating 1 mg Oxy133 dissolved in ethanol; collagen sponge incorporating ethanol only; collagen sponge incorporating 75 μg BMP-2; or no treatment. A minimum of six defects for each experimental condition was studied. The wounds were carefully closed with absorbable suture and a postoperative analgesic was administered for 3 days. Six weeks after implantation, the animals were sacrificed and the entire calvarium was harvested. The specimens were immediately fixed in 10% buffered formalin. The fixed tissue specimens were first examined by micro-computed tomography (micro-CT; mCT40, Scanco Medical, Bassersdorf, Switzerland) at an X-ray energy level of 55 kVp with a current of 72 mA. Threedimensional (3D) images of the cranial bones, including the areas of the reconstructed defects, were rendered and calculated, using a customized computational program, by digitally extracting the bone image using the predetermined threshold of 220. Micro-CT images were then scored for bone regeneration by three blinded and independent reviewers, using a previously described grading scale (Patel et al., 2008; Spicer et al., 2012) that accounts for the extent of bony projections as a measure of overall bone growth. Briefly, the scoring for extent of bony bridging and union was: score 0, no bone formation within defect; score 1, few bony spicules dispersed through defect; score 2, bony J Tissue Eng Regen Med (2015) DOI: 10.1002/term
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bridging only at defect borders; score 3, bony bridging over partial length of defect; and score 4, bony bridging within the entire span of the defect at its longest point.
2.8. Inhibition of Oxy133-mediated in vitro osteogenesis with the Hh signalling inhibitor cyclopamine The Hh signalling pathway has been shown to play a role in the process of osteogenesis in murine cells (Dwyer et al., 2007; Kim et al., 2007) and rabbit cells (Hokugo et al., 2013a, 2013b). In order to determine whether Hh signalling plays a role in the mechanism by which oxysterols modulate osteogenesis, cell cultures were treated with cyclopamine, a known inhibitor of the Hh pathway, prior to treatment with Oxy133. Rabbit BMSCs were pretreated for 2 h with control vehicle (DMSO) or cyclopamine at concentrations of 2 or 4 μM. Following pretreatment, 1 μM Oxy133 or control vehicle was added to cell cultures. After 4 days of incubation, ALP activity assays were performed as described above.
2.9. Inhibition of Oxy133-mediated in vivo bone regeneration with cyclopamine In order to determine whether Hh signalling plays a role in the mechanism by which Oxy133 modulates in vivo bone regeneration, we added 2 mg cyclopamine dissolved in ethanol to collagen sponges incorporating 10 mg (high dose) Oxy133 and implanted these into calvarial bone defects. Collagen sponges containing only 2 mg cyclopamine dissolved in ethanol were also implanted into separate defects to serve as controls. Six weeks after implantation, bone regeneration assessments were performed as described above.
2.10. Statistical analysis All in vitro experiments were repeated a minimum of three times. Statistical analyses were performed by oneway ANOVA with Dunnett’s multiple comparison of means tests; p < 0.05 was considered statistically significant.
3. Results 3.1. Effect of Oxy133 on rabbit BMSCs The osteogenic response of rBMSCs treated with osteogenic medium to Oxy133 was first evaluated by measurement of subsequent ALP activity levels. ALP activity was significantly elevated in rBMSCs exposed to Oxy133 and 50 ng/ml BMP-2 in comparison to control cells treated with vehicle alone. Oxy133 induced a greater response than 50 ng/ml BMP2 (Figure 2). Copyright © 2015 John Wiley & Sons, Ltd.
Figure 2. Rabbit BMSCs at confluence were treated with control medium (CM) or Oxy133 in osteogenic medium (OM). Positive control cultures were treated with 50 ng/ml BMP-2, known to activate ALP activity. After 4 days of incubation, ALP activity was determined in cell homogenates by a colorimetric assay. Data from a representative experiments are reported as the mean ± SD of quadruplicate determinations, normalized to protein concentration; *p < 0.05 vs CM
In order to further determine the effects of Oxy133 on osteogenic function in rBMSCs, we evaluated the ability of treated cells to form a mineralized matrix, using alizarin red staining. Cell cultures were maintained for 21 days and then assessed with alizarin red. Cells cultured in nonosteogenic medium (CM) demonstrated a negligible mineralization response, whereas areas of positive alizarin red staining were observed in cultures of cells treated with OM, Oxy133 and BMP-2. A significant increase in mineralization relative to OM cultures was noted in cells treated with Oxy133, as well as in cultures treated with BMP-2 (Figure 3A). In order to make a quantitative assessment of mineralization response, a 45Ca incorporation assay was performed. The amount of incorporated 45Ca was significantly higher in cultures treated with Oxy133 than that seen in controls and in cells treated with BMP2 (Figure 3B). In order to determine the effect of Oxy133 on the expression of genes of osteogenic differentiation in rBMSCs, the mRNA expression of key markers of osteogenesis, including RUNX2, COL and OSX, was examined. Following 4 days of treatment with OM supplemented with either Oxy133 or 50 ng/ml BMP-2, real-time PCR was performed. Treatment of rBMSCs with Oxy133 resulted in a significant increase in mRNA expression of all three gene markers relative to control, and this increase was either equivalent to or greater than the induction observed in cells treated with BMP-2 (Figure 4).
3.2. Bone regeneration in rabbit cranial bone defects To demonstrate the potential clinical relevance of these positive in vitro results, we used Oxy133 as a means of enhancing in vivo bone regeneration in an established J Tissue Eng Regen Med (2015) DOI: 10.1002/term
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in animals treated with collagen sponge containing control vehicle only. By comparison, robust bone regeneration was observed in defects treated with collagen sponges incorporating either Oxy133 or BMP-2. Low-dose (1 mg) Oxy133 yielded substantial bone deposition, with some thin and immature bone centrally. However, complete bone healing was noted in specimens treated with high-dose (10 mg) Oxy133. These results were equivalent to those observed in defects treated with 75 μg BMP-2 (Figure 5A). When examined under higher magnification, mature lamellar bone (similar to naïve bone tissue) was observed within defects treated with collagen sponges incorporating either Oxy133 or BMP-2. It is important to note that there were no inflammatory cells in the vicinity of the regenerated bone (Figure 5B). Figure 5C shows the results of this scoring for regenerated bone at defects treated with various implants. Collagen sponges incorporating Oxy133 (10 mg) and collagen sponges incorporating BMP-2 received significantly higher scores for union compared to collagen sponge-only groups.
3.3. In vitro and in vivo inhibition of Oxy133mediated osteogenesis with cyclopamine
Figure 3. (A) Alizarin red staining for calcium mineralization in rBMSCs after 21 days of culture with control medium (CM), osteogenic medium (OM) and OM with 1 μM Oxy133 or 50 ng/ml BMP-2: representative images of alizarin red staining are shown; pink/red areas, background areas of calcium mineralization; black areas, dense mineral deposition. (B) Minerali45 zation was also quantified using a Ca incorporation assay; *p < 0.05 vs CM
animal model for craniofacial reconstruction. Histological and radiographic analyses of calvarial defect specimens harvested 6 weeks after implant placement revealed a persistent full-thickness defect in animals treated with no reconstruction. Some peripheral bone regrowth was noted
The role of the Hh signalling pathway in the osteoinductive activity of Oxy133 was assessed through Hh pathway inhibition in vitro and in vivo. Addition of 2 or 4 μM cyclopamine to rBMSC cell cultures consistently reversed the increases in ALP activity noted in cells treated with Oxy133; these doses were chosen based on unpublished data from our group, demonstrating the antagonistic role of cyclopamine in BMP-2-induced spinal fusion in rats. With cylcopamine treatment, ALP activity was decreased to a level similar to that seen in cells treated with OM only (Figure 6A). When cyclopamine was added to collagen sponges carrying 10 mg Oxy133 and implanted in calvarial defects, micro-CT and histology demonstrated significant attenuation of the bone regeneration originally noted in defects treated with Oxy133 (Figure 5). Rather than robust bone deposition and complete defect healing, new bone was
Figure 4. Oxy133 induces expression of markers of osteogenic differentiation in rBMSCs: BMSCs cultured to confluence in control medium (CM), osteogenic medium (OM), OM with 1 μM Oxy133 or OM with 50 ng/ml of BMP-2. After 4 days in culture, mRNA expression was measured by quantitative real-time PCR. Data from representative experiments are reported as mean ± SD of triplicate determinations; *p < 0.05 vs CM Copyright © 2015 John Wiley & Sons, Ltd.
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Figure 5. (A) (Right) 3D renderings and (left) histological images (H&E staining) of rabbit cranial bone defects, 6 weeks after undergoing no reconstructive treatment (Defect) or after implantation with collagen sponge carrying control vehicle only (CS), collagen sponge incorporating 1 mg Oxy133 [CS + Oxy133 (1 mg)], collagen sponge incorporating 10 mg Oxy133 [CS + Oxy133 (10 mg)] or collagen sponge incorporating 75 μg BMP-2 (CS + BMP-2); bar = 1 mm. (B) Higher magnification of histological findings of the defect treated with collagen sponge incorporating 10 mg Oxy133 or BMP-2; bar = 100 μM. (C) Scoring for bony bridging and union within the defect treated with various implants; *p < 0.05 vs CS
only seen peripherally and central full-thickness defects persisted (Figure 6B). The results of the scoring for regenerated bone further supported this observation: defects Copyright © 2015 John Wiley & Sons, Ltd.
implanted with 10 mg Oxy133 plus cyclopamine received significantly lower scores than defects treated with 10 mg Oxy133 alone (Figure 6C). J Tissue Eng Regen Med (2015) DOI: 10.1002/term
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Figure 6. (A) ALP activity is diminished by addition of cyclopamine (Cyc): rabbit BMSCs at confluence were pretreated with control vehicle (2 μM) or Cyc (4 μM) for 2 h; next, 1 μM Oxy133 was added to cell cultures; ALP activity was measured after 4 days; data are normalized to ALP activity in cells treated with CM; Tukey–Kramer multiple comparison test; *significant differences (p < 0.05) compared to Oxy133 1 μM. (B) (Left) 3D renderings and (right) histological images of rabbit cranial bone defects, 6 weeks after implantation with a collagen sponge incorporating 10 mg Oxy133 and 2 mg Cyc or collagen sponge incorporating 2 mg Cyc alone; bar = 1 mm. (C) Scoring for bony bridging and union within the defect treated with various implants; *p < 0.05 vs CS + 10 mg Oxy133
4. Discussion Among the growth factors that are effective in stimulating bone regeneration, BMPs are well-established and powerful osteoinductive agents (Urist, 1965). BMPs strongly induce osteogenic differentiation of mesenchymal stem cells and osteoblastic progenitor cells. Because of these qualities, BMPs have been central to the development of clinical strategies aimed at generating a viable bone graft substitute for use in the clinical setting. Commercial products incorporating recombinant human (rh)BMP-2 (InfuseTM, Medtronic, Minneapolis, MN, USA) and rhBMP7 (OP-1TM, Stryker Biotech, Hopkinton, MA, USA) have been approved for clinical use by the US Food and Drug Administration (FDA) in spine and maxillofacial surgery (Cahill et al., 2011). Numerous ’off-label’ clinical uses of BMP-2 have also been reported (Glied and Kraut, 2010; Copyright © 2015 John Wiley & Sons, Ltd.
Ong et al., 2010). However, the high concentrations of BMP used in these products have been associated with severe deleterious side-effects, including acute airway obstruction (Yaremchuk et al., 2010a, 2010b), heterotopic bone formation (Brower and Vickroy, 2008), sterile seroma (Garrett et al., 2010) and profound soft tissue oedema (Shah et al., 2008), prompting the FDA to issue a formal warning regarding these complications, which may be potentially life threatening. Moreover, these products are expensive and may cost more to implement in clinical therapies than traditional bone autografts (Buttermann, 2008). BMPs are further limited by their non-specific activity: they induce not only osteogenic but also adipogenic differentiation of mesenchymal stem cells by induction of PPARγ expression in vitro and in vivo (Kang et al., 2009). The quality of regenerated bone may therefore be suboptimal. Finally, rhBMPs are not approved for use in skeletally immature patients and therefore are not applicable to the treatment of paediatric patients, who account for a large number of the complex congenital craniofacial reconstructive cases that present to tertiary care referral centres. Considering the potential drawbacks associated with BMP-2 therapy, searching for other factors that can be used as alternatives to, or in conjunction with, BMP-2 to minimize the disadvantages associated with its use is a very worthwhile endeavour. To that end, we have studied oxysterols for their potential use in clinical bone regeneration therapies. Oxysterols comprise a large family of 27-carbon molecules that are oxygenated derivatives of cholesterol. They are naturally present in the vascular system and tissues of humans and other higher animals (Bjorkhem et al., 2002) and are involved in diverse biological processes, including cholesterol homeostasis, sphingolipid metabolism, platelet aggregation and apoptosis (Kha et al., 2004). We have found that oxysterols show promising bioactivity in bone tissue engineering (Amantea et al., 2008; Kha et al., 2004). We originally showed that a specific naturally occurring oxysterol, 20(S)-hydroxycholesterol (Figure 1, left), induces the osteogenic differentiation of murine mesenchymal stem cells while simultaneously inhibiting adipogenesis (Kha et al., 2004). From a series of structure– activity relationship studies, we further observed that the hydroxyl group on C20 of the side chain of 20(S)hydroxycholesterol is essential for activation of Hh signalling and induction of osteogenesis. We next described how Oxy49, a novel oxysterol analogue of 20 (S)-hydroxycholesterol (Figure 1, centre), induces osteogenesis in rBMSCs in vitro more robustly than previously described oxysterols (Hokugo et al., 2013a, 2013b). Most recently, we have synthesized another oxysterol analogue, Oxy133 (Figure 1, right), that has the highest osteoinductive potency observed to date, is more easily and inexpensively synthesized than other analogues and might therefore serve as a better candidate for large-scale development geared toward clinical translation (Montgomery et al., 2014). In this study we demonstrated the osteoinductive efficacy of Oxy133 both in vitro and in vivo. ALP activity J Tissue Eng Regen Med (2015) DOI: 10.1002/term
A. Li et al.
was measured in cells that were cultured in CM and OM supplemented with various concentrations of Oxy133 and compared to cells treated witb BMP-2. The dose of BMP-2 used in this study was 50 ng/ml, which has previously been shown to induce ALP activity in rBMSCs (Roostaeian et al., 2006). ALP enzymatic activity is indicative of successful synthesis of a functional ALP protein, which suggests differentiation along an osteogenic pathway (Mornet et al., 2001; Siffert, 1951). The induction of ALP activity in rBMSCs by 1 μM Oxy133 was greater than the activity induced by BMP-2. This observation demonstrates the potent osteoinductive activity of Oxy133 in this in vitro rBMSC system and is consistent with similar results found in murine MSC cultures treated with oxysterol (Kha et al., 2004; Mackie, 2003). We also included a comparison of the effects of Oxy133 to Oxy49 to evaluate its osteoinductive activity relative to that of the earlier generation oxysterol analogue, which we have previously shown has potent in vitro and in vivo osteogenic effects (Montgomery et al., 2014). Oxy133 induced a significantly greater amount of ALP activity compared to Oxy49, confirming its greater osteognenic potential. Mineral deposition begins during extracellular matrix maturation, and is an indicator of the late stages of osteogenic differentiation (Kim et al., 2007; Mornet et al., 2001). Mineralization assays, therefore, not only provide confirmation of cellular commitment to an osteoblastic differentiation pathway but also provide an assessment of the functionality of the mature cell (Lizard et al., 1996). In this study, cells treated with 1 μM Oxy133 showed a significantly increased capacity for mineralization compared to controls, and compared to cells treated with BMP-2. We also examined the effect of Oxy133 on the expression of genes associated with osteogenic differentiation. RUNX2 expression is indicative of the early commitment of MSCs to osteochondrogenic progenitors, whereas terminal differentiation is linked to the expression of OSX (Satija et al., 2007). The majority of the organic matrix of bone is made up of COL1 (Lee et al., 2007), the expression of which has previously been found to occur at high levels in the early stages of osteogenesis (Denhardt et al., 2001). We have demonstrated that 1 μM Oxy133 consistently and significantly increases mRNA expression of all these markers in rBMSCs. These results, along with our ALP activity data, collectively suggest that, at an ideal concentration of 1 μM, Oxy133 is able to induce in vitro differentiation of rBMSCs along an osteogenic pathway. This study used 8 mm calvarial defects to evaluate bone regeneration in skeletally mature rabbits. Although the 8 mm defect used in this study is smaller than the 15 mm rabbit cranial defect favoured by some authors (Hollinger and Kleinschmidt, 1990), healing was very limited in the absence of any treatment, indicating that the 8 mm defect size was incapable of complete spontaneous healing at week 6. Previous studies have also reported incomplete spontaneous healing in 5 mm (Hokugo et al., 2005; Meikle et al., 1994) and 6 mm (Hokugo et al., 2013a, Copyright © 2015 John Wiley & Sons, Ltd.
2013b) calvarial defects in rabbits, suggesting that an 8 mm cranial bone defect is an appropriate model to assess the regenerative capacity of an osteoinductive agent. In our model, we did not observe radiographic or histological evidence of complete bone regeneration in defects that were left untreated. Defects treated with collagen sponges incorporating a control vehicle demonstrated very limited amounts of peripheral bone formation on micro-CT compared to the defects with sponges containing Oxy133 or BMP-2. By comparison, bone regeneration was clearly observed in defects implanted with sponges containing Oxy133 or BMP-2. Low-dose Oxy133 yielded incomplete bone healing, while the higher dose resulted in complete healing of the defect. The bones of the craniofacial skeleton develop via intramembranous ossification through condensation of mesenchymal tissue into vascularized connective tissue and subsequent mineralization (Sadler and Langman, 2012). This process is mediated by various biochemical signals, including parathyroid hormone-related protein (PTHrP), vascular endothelial growth factor (VEGF), matrix metalloproteinase 13 (MMP13), Noggin and BMP-2 (Mackie et al., 2011). We have previously observed the induction of robust osteogenic responses by Oxy133 in murine embryonic fibroblasts and marrow stromal cells, as well as in primary human mesenchymal stem cells (Montgomery et al., 2014). Given these findings, it is possible that in our in vivo defect model, both undifferentiated mesenchymal stem cells that accumulate at the site of injury and resident calvarial preosteoblasts within the adjacent periosteum, are induced to differentiate along osteogenic pathways under the influence of Oxy133, perhaps mimicking the role of BMP-2 in the process of intramembranous ossification. An understanding of the mechanisms by which this process unfolds is integral to understanding it more fully and developing its translational potential. Hh signalling has been noted to play a role in the healing of cranial bone. Specifically, Indian Hh is the critical factor in intramembranous calvarial ossification (Lenton et al., 2011), while Sonic Hh is a well known potent osteogenic factor in various cell types and experimental model systems (Abzhanov et al., 2007). The activation of the Hh signalling pathway by oxysterols potentially influences intramembranous bone formation, and this warrants evaluation. Our in vitro results show that when oxysteroltreated rBMSCs are exposed to cyclopamine, a known inhibitor of the Hh signalling pathway, ALP activity is significantly decreased (Figure 6A). We have observed this phenomenon with two distinct analogues of oxysterol, Oxy133 and Oxy49. Our findings indicate that Hh signalling plays a role in Oxy133-mediated osteogenesis in rBMSCs. In vivo, we noted significant attenuation of Oxy133-induced bone healing in calvarial defects treated with both Oxy133 and cyclopamine, again suggesting that Hh not only plays a role in the in vitro processes that drive osteogenesis, but that its role also has potential clinical significance (Figure 6B, C). Our studies clearly demonstrate that Oxy133, and in turn activation of Hh J Tissue Eng Regen Med (2015) DOI: 10.1002/term
Oxy133 promotes bone regeneration
signalling, appear to induce osteogenesis in the context of both intramembrenous bone formation (calvarial bone regeneration) and endochondral bone formation (spinal fusion) (Montgomery et al., 2014) that we have previously reported. Further study is needed to fully elucidate the relationship between oxysterols and Hh in bone regeneration.
5. Conclusions Oxysterols represent a readily available, naturally occurring, inert and relatively inexpensive material upon which a tissue-engineering solution to an elusive bone graft substitute can potentially be based. Semi-synthetic oxysterol analogues have been shown to support osteogenesis. We have demonstrated that Oxy133, a novel analogue of naturally occurring oxysterols, has potent osteoinductive capacity in both in vitro and in vivo models. Moreover, we have initial insights into the mechanism by which it exerts its osteogenic effects. Oxysterols have the potential to help us to address complex craniofacial defects in a manner that minimizes costs and morbidity without compromising patient outcomes. Further investigation
into the translational applicability of this class of compounds is currently under way.
Conflict of interest As the Scientific Founder of MAX BioPharma, Dr Parhami has a financial interest in Oxy133, which has been licensed by the company from UCLA for further development. Dr Stappenbeck is Director of Chemistry at MAX BioPharma and therefore has financial interests in the technology used in this study.
Acknowledgements The authors are especially grateful for contributions to animal experiments from Dr Wei Zhou, Department of Anesthesiology, David Geffen School of Medicine at UCLA. This study was supported by grants from the Annenberg Foundation for Craniofacial Surgery and Research at the University of California Los Angeles, Komedyplast Craniofacial Research, the Cleft Plate Foundation/Craniofacial Anomalies Research and the PSF National Endowment for Plastic Surgery.
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J Tissue Eng Regen Med (2015) DOI: 10.1002/term
