Ectopic Bone Growth Research Articles

Complications and Revision Surgery of Forearm Fractures.

Nonunion or malunion of forearm fractures may result in restricted range of motion or persistent pain, leading to limitations in activities of daily living. Complications may also present in the form of ectopic bone growth or persistent infections. A systematic method to evaluate complications resulting from forearm fractures is helpful to both determine the cause for the failure and plan for adequate reconstruction.

Regenerative engineering of long bones using the small molecule forskolin

Bone grafting procedures have become increasingly common in the United States, with approximately 500,000 cases occurring each year at a societal cost exceeding $2.4 billion. Recombinant human bone morphogenetic proteins (rhBMPs) are therapeutic agents that have been widely used by orthopedic surgeons to stimulate bone tissue formation alone and when paired with biomaterials. However, significant limitations such as immunogenicity, high production cost, and ectopic bone growth from these therapies remain. Therefore, efforts have been made to discover and repurpose osteoinductive small-molecule therapeutics to promote bone regeneration. Previously, we have demonstrated that a single-dose treatment with the small-molecule forskolin for just 24 h induces osteogenic differentiation of rabbit bone marrow-derived stem cells invitro, while mitigating adverse side effects attributed with prolonged small-molecule treatment schemes. In this study, we engineered a composite fibrin-PLGA [poly(lactide-co-glycolide)]-sintered microsphere scaffold for the localized, short-term delivery of the osteoinductive small molecule, forskolin. Invitro characterization studies showed that forskolin released out of the fibrin gel within the first 24 h and retained its bioactivity toward osteogenic differentiation of bone marrow-derived stem cells. The forskolin-loaded fibrin-PLGA scaffold was also able to guide bone formation in a 3-mo rabbit radial critical-sized defect model comparable to recombinant human bone morphogenetic protein-2 (rhBMP-2) treatment, as demonstrated through histological and mechanical evaluation, with minimal systemic off-target side effects. Together, these results demonstrate the successful application of an innovative small-molecule treatment approach within long bone critical-sized defects.

Two-phase matrices for the controlled release of therapeutic proteins.

Controlled and sustained delivery of therapeutic proteins is crucial for achieving desired effects in wound healing applications. Yet, this remains a challenge in growth factor delivery for bone tissue engineering. Current delivery systems can lead to negative side effects, such as ectopic bone growth and cancer, due to the over administration of growth inducing proteins. Here, we have developed a two-phase system for the controlled release of therapeutic proteins. The system consists of protein-loaded poly(methacrylic acid)-based nanoparticles conjugated to chitosan scaffolds. The effect of co-monomer hydrophilicity and crosslinking density on nanoparticle properties was evaluated. It was found that the release kinetics of model therapeutic proteins were dependent on nanoparticle hydrophilicity. The chitosan scaffold, chosen for its biocompatibility and osteogenic properties, provided additional barriers to diffusion and promoted nanoparticle retention, leading to more sustained protein delivery. Additionally, the ability of MC3T3-E1 pre-osteoblast cells to proliferate on scaffolds with and without conjugated nanoparticles was evaluated and all scaffolds were shown to promote cell growth. The results demonstrate that the two-phase scaffold system presents a superior strategy for the sustained and controlled release of therapeutic proteins for bone tissue engineering applications.

Medical Malpractice Litigation Due to Off-Label Use of Bone Morphogenetic Protein.

Retrospective cohort study. To analyze the incidence and characteristics of malpractice lawsuits pertaining to the off-label use of Bone morphogenetic protein (BMP). BMP continues to be a popular alternative to the use of autologous bone graft during spinal fusion procedures. However, ~85% of BMP is used in an off-label manner, which may expose surgeons to claims of malpractice. Westlaw and VerdictSearch were queried for malpractice claims filed between 2000 and 2022 using the keywords "bone graft" and "spine." Case inclusion criteria were defined as a plaintiff's basis of litigation resting on a claim of medical malpractice due to off-label use of BMP. Additional collected data included the date of the case hearing, plaintiff's sex and age, defendants named in the lawsuit, verdict ruling, location of the filed claim, payment or settlement amount, sustained injuries, and additional allegations. Of 971 claims reviewed, 89 cases were due to off-label BMP use. The posterior approach was identified as the most common approach among the 89 cases. Of the 30 cases naming a surgeon defendant, 50% included allegations of insufficient informed consent. The most frequent adverse events were ectopic bone growth resulting in chronic pain or nerve damage. Zero cases involved heterotopic ossification, wound dehiscence, graft subsidence, hematoma, bladder retention, or retrograde ejaculation. A clear understanding that on-label BMP use is specific to single-level, anterior or anterolateral approaches between L2-S1 with vendor-specific cages is crucial for mitigating malpractice disputes. Though off-label use of BMP may incite litigation, the findings suggest court rulings are favorable for defendants as zero cases, resulting in plaintiff verdicts. Nevertheless, surgeons should balance the potential benefits of off-label use of BMP with the increased risk of litigation, and it may be advisable to disclose the use of BMP, whether on-label or off-label, in the informed consent.

TrkA+ Neurons Induce Pathologic Regeneration After Soft Tissue Trauma.

Heterotopic ossification (HO) is a dynamic, complex pathologic process that often occurs after severe polytrauma trauma, resulting in an abnormal mesenchymal stem cell differentiation leading to ectopic bone growth in soft-tissues including tendons, ligaments, and muscles. The abnormal bone structure and location induce pain and loss of mobility. Recently, we observed that NGF (Nerve growth factor)-responsive TrkA (Tropomyosin receptor kinase A)-expressing nerves invade sites of soft-tissue trauma, and this is a necessary feature for heterotopic bone formation at sites of injury. Here, we assayed the effects of the partial TrkA agonist Gambogic amide (GA) in peritendinous heterotopic bone after extremity trauma. Mice underwent HO induction using the burn/tenotomy model with or without systemic treatment with GA, followed by an examination of the injury site via radiographic imaging, histology, and immunohistochemistry. Single-cell RNA Sequencing confirmed an increase in neurotrophin signaling activity after HO-inducing extremity trauma. Next, TrkA agonism led to injury site hyper-innervation, more brisk expression of cartilage antigens within the injured tendon, and a shift from FGF to TGFβ signaling activity among injury site cells. Nine weeks after injury, this culminated in higher overall levels of heterotopic bone among GA-treated animals. In summary, these studies further link injury site hyper-innervation with increased vascular ingrowth and ultimately heterotopic bone after trauma. In the future, modulation of TrkA signaling may represent a potent means to prevent the trauma-induced heterotopic bone formation and improve tissue regeneration.

Developing a combat-relevant translatable large animal model of heterotopic ossification

Heterotopic ossification (HO) refers to ectopic bone formation, typically in residual limbs following trauma and injury. A review of injuries from Operation Iraqi Freedom (OIF) and Operation Enduring Freedom (OEF) indicated that approximately 70% of war wounds involved the musculoskeletal system, largely in part from the use of improvised explosive devices (IED) and rocket-propelled grenades (RPG). HO is reported to occur in approximately 63%–65% of wounded warriors from OIF and OEF. Symptomatic HO may delay rehabilitation regimens since it often requires modifications to prosthetic limb componentry and socket size. There is limited evidence indicating a mechanism for preventing HO. This may be due to inadequate models, which do not produce HO bone structure that is morphologically similar to HO samples obtained from wounded warfighters injured in theatre. We hypothesized that using a high-power blast of air (shockwave) and simulated battlefield trauma (i.e. bone damage, tourniquet, bacteria, negative pressure wound therapy) in a large animal model, HO would form and have similar morphology to ectopic bone observed in clinical samples. Initial radiographic and micro-computed tomography (CT) data demonstrated ectopic bone growth in sheep 24 weeks post-procedure. Advanced histological and backscatter electron (BSE) analyses showed that 5 out of 8 (63%) sheep produced HO with similar morphology to clinical samples. We conclude that not all ectopic bone observed by radiograph or micro-CT in animal models is HO. Advanced histological and BSE analyses may improve confirmation of HO presence and morphology, which we demonstrated can be produced in a large animal model.

3505 Fracture Targeted Parathyroid Hormone Agonist As An Effective Pharmaceutical For Bone Repair in Mouse and Canine Models

OBJECTIVES/SPECIFIC AIMS: The primary objective of this study was to evaluate the performance of a bone fracture targeted systemically administrable bone anabolic as a potential therapeutic for bone fracture repair. Currently all bone fracture repair therapeutic require local administration during surgery. However, the population that need the most assistance in repair bone fractures are not eligible for surgery. So, it was our goal to design an inject-able therapeutic to assist in bone fracture repair to reduce the invasiveness. The injectable nature of it allows for repair administration of the bone anabolic and for therapeutic effect throughout the entire bone fracture healing process. Targeting it to the bone fracture site reduces the toxicity and increases the efficacy. METHODS/STUDY POPULATION: METHODS To achieve the above objective, a bone mineral-(hydroxyapatite-) targeting oligopeptide was conjugated to the non-signaling end of an engineered parathyroid hormone related protein fragment 1-46 with substitutions at Glu22,25, Leu23,28,31, Aib29, Lys26,30 (ePTHrP). The negatively charged oligopeptide has been shown to target raw hydroxyapatite with remarkable specificity, while the attached PTHrP has been demonstrated to induce sustained and accelerated bone growth under control of endogenous morphogenic regulatory factors. The conjugate’s specificity arises from the fact that raw hydroxyapatite is only exposed whenever a bone is fractured, surgically cut, grafted, or induced to undergo accelerated remodeling. The hydroxyapatite-targeted conjugate can therefore be administered systemically (i.e. without invasive surgery or localized injection) and still accumulate on the exposed hydroxyapatite at the fracture site where it accelerates the healing process Murine in vivo experiments were conducted on female Swiss Webster mice (10 per group). Femoral fractures were induced with a 3-point bending device and stabilized. Mice were dosed with 3 nmol/kg/d of targeted-ePTHrP, non-conjugated (free) ePTHrP, or saline. Following a 4-week study, fracture callus densities were measured using microCT. Canine in vivo experiments were conducted on 1-year-old male beagles. Beagles underwent a 10 mm bilateral ulnar ostectomy. Two dogs in the treatment group and Three dogs in the control group were dosed daily with either targeted-ePTHrP 0.5nmol/kg/d or saline respectively. Dogs were x-rayed weekly for the first 6 weeks and then every other week thereafter. One tailed ANOVA followed by Dunnett’s post-hoc test was used to establish significance. All animal experiments were conducted as described in approved IACUC protocols. P<0.05 was considered significant. RESULTS/ANTICIPATED RESULTS: RESULTS SECTION: In the murine studies we observed a marked increase in fracture callus size and a 2-fold increase in bone deposition was observed in the targeted-ePTHrP group over the saline group (P<0.01). A significant doubling in bone density was also observed. Targeted-ePTHrP group fractured femurs were able to achieve their pre-fracture strength as early as 3 weeks compared to 9 weeks in the saline mice representing a 66% reduction in healing time. In the canine studies, we observe a significantly higher closure of the ostectomy gap than saline controls (P<0.05). In addition, no significant differences in weight are observed in the treatment vs. saline controls. No significant difference between the control group and treatment groups was found in a histological investigation of the organs. DISCUSSION/SIGNIFICANCE OF IMPACT: DISCUSSION: Although attempts have been made in developing a systemically administered fracture therapeutic for fracture repair, i.e. teriparatide, to date, no such anabolics have been approved for this use. In these studies there is evidence that anabolic activity was occurring at the fracture site, but at a level that did not meet FDA required end-points.2 It is plausible that if sufficient drug were to be delivered to a fracture site then improved fracture repair would be possible. In previous studies, we demonstrated fracture specific accumulation bone anabolics can be achieved by modifying the drug with acidic oligopeptides.3 Here, by modifying a safe, clinically proven, parathyroid hormone receptor agonist with an acidic oligopeptide we observe improved bone deposition and strength in mice. Furthermore, when administered to canine critical sized defect ostectomies, a more relevant and difficult model, we observe improved ostectomy closure. CLINICAL RELEVANCE:: The ability to accelerate bone fracture repair is a fundamental need that has not been addressed by conventional methods. By targeting bone anabolic agents to bone fractures, we can deliver sufficient concentrations of anabolic agent to the fracture site to accelerate healing, thus avoiding surgery and any ectopic bone growth associated with locally-applied bone anabolic agents.

A requirement for Fgfr2 in middle ear development.

The skeletal structure of the mammalian middle ear, which is composed of three endochondral ossicles suspended within a membranous air-filled capsule, plays a critical role in conducting sound. Gene mutations that alter skeletal development in the middle ear result in auditory impairment. Mutations in fibroblast growth factor receptor 2 (FGFR2), an important regulator of endochondral and intramembranous bone formation, cause a spectrum of congenital skeletal disorders featuring conductive hearing loss. Although the middle ear malformations in multiple FGFR2 gain-of-function disorders are clinically characterized, those in the FGFR2 loss-of-function disorder lacrimo-auriculo-dento-digital (LADD) syndrome are relatively undescribed. To better understand conductive hearing loss in LADD, we examined the middle ear skeleton of mice with conditional loss of Fgfr2. We find that decreased auditory function in Fgfr2 mutant mice correlates with hypoplasia of the auditory bulla and ectopic bone growth at sites of tendon/ligament attachment. We show that ectopic bone associated with the intra-articular ligaments of the incudomalleal joint is derived from Scx-expressing cells and preceded by decreased expression of the joint progenitor marker Gdf5. Together, these results identify a role for Fgfr2 in development of the middle ear skeletal tissues and suggest potential causes for conductive hearing loss in LADD syndrome.

Biomaterials for the Delivery of Growth Factors and Other Therapeutic Agents in Tissue Engineering Approaches to Bone Regeneration.

Bone fracture followed by delayed or non-union typically requires bone graft intervention. Autologous bone grafts remain the clinical “gold standard”. Recently, synthetic bone grafts such as Medtronic's Infuse Bone Graft have opened the possibility to pharmacological and tissue engineering strategies to bone repair following fracture. This clinically-available strategy uses an absorbable collagen sponge as a carrier material for recombinant human bone morphogenetic protein 2 (rhBMP-2) and a similar strategy has been employed by Stryker with BMP-7, also known as osteogenic protein-1 (OP-1). A key advantage to this approach is its “off-the-shelf” nature, but there are clear drawbacks to these products such as edema, inflammation, and ectopic bone growth. While there are clinical challenges associated with a lack of controlled release of rhBMP-2 and OP-1, these are among the first clinical examples to wed understanding of biological principles with biochemical production of proteins and pharmacological principles to promote tissue regeneration (known as regenerative pharmacology). After considering the clinical challenges with such synthetic bone grafts, this review considers the various biomaterial carriers under investigation to promote bone regeneration. This is followed by a survey of the literature where various pharmacological approaches and molecular targets are considered as future strategies to promote more rapid and mature bone regeneration. From the review, it should be clear that pharmacological understanding is a key aspect to developing these strategies.

Effect of poly-L-co-D,L-lactide scaffolds combined with simvastatin and mesenchymal cells on calvarial bone defects and ectopic subcutaneous bone formation

Event Abstract Back to Event Effect of poly-L-co-D,L-lactide scaffolds combined with simvastatin and mesenchymal cells on calvarial bone defects and ectopic subcutaneous bone formation Stephanie V. Edelmuth1*, Kennedy S. Ramos1*, Khatharine S. Aoki1*, Karlla K. Gaiotto1*, Bruna A. Más2*, Mona L. Barbo3*, Eliana A. Duek4* and Newton M. Oliveira3* 1 Pontifical Catholic University of São Paulo, Medical student, Faculty of Medical Sciences and Health, Brazil 2 Pontifical Catholic University of São Paulo, Laboratory of Biomaterials, Faculty of Medical Sciences and Health, Brazil 3 Pontifical Catholic University of São Paulo, Morphology and Pathology Department, Faculty of Medical Sciences and Health, Brazil 4 Pontifical Catholic University of São Paulo, Physiology Department, Faculty of Medical Sciences and Health, Brazil Tissue bioengineering has become popular as a method of repair of damaged tissues, and allows for both an aesthetic and functional result[1]. One of the biomaterials used in the treatment of bone defects are the hydrolytically degradable polymers, specifically the poly-L-co-D,L-lactide (PLDLA)[2]. PLDLA is considered an attractive material for drug delivery systems, such as for simvastatin release. Several studies show that statins have the capacity of stimulating bone formation by increasing expression of bone morphogenetic protein-2 (BMP-2)[3]. Mesenchymal stem cells (MSC) are also involved in the process of bone growth, since they have potential to differentiate into osteoblasts and osteocytes[4]. Based on these concepts, this study analyzed the effect of PLDLA scaffolds with simvastatin and MSC in regards to their capacity to promote bone formation in both calvarial defects and the subcutaneous tissues of Wistar rats[5]. The 98 animals used for the study were divided according to the area of scaffold implantation, time of euthanasia and treatment applied. The implant areas were collected post-euthanasia and submitted for histologic analysis. New bone formation was detected in the animals who received calvarial implants, with statistical significance. In comparison to negative control, new bone tissue was especially observed in the groups receiving simvastatin and MSC (p < 0.01). No ectopic bone growth was seen in the subcutaneous tissue. The bio-affinity of the polymeric membrane and the osteogenic character of simvastatin and MSC were confirmed in this study.

Collagen-PEG composite microspheres for bone regeneration

Event Abstract Back to Event Collagen-PEG composite microspheres for bone regeneration Candice Sears1, Sarah Dang2, Colin Dodson1, Allison Rice-Ficht3, Carl Gregory3 and Roland Kaunas1 1 Texas A&M University, Biomedical Engineering, United States 2 Texas A&M University, Chemical Engineering, United States 3 Texas A&M Health Science Center, Molecular and Cellular Medicine, United States Introduction: Of the 5.6 million bone fractures that occur annually in the United States, about 10% fail to repair[1]. The canonical Wingless (cWnt) signaling pathway is critical for healing of bone fractures[2]. We have demonstrated that inhibiting peroxisome proliferator-activating receptor gamma with GW9662 (GW) reduces negative cross-talk on the cWnt pathway, resulting in a pro-osteogenic hMSC phenotype (OEhMSCs)[3]. OEhMSCs secrete an extracellular matrix (hMatrix) that mimics the composition of anabolic bone tissue and strongly enhances hMSC retention and subsequent bone repair in vivo[3]. We designed composite microspheres for co-administration of GW and hMatrix to facilitate cell retention and bone repair in vivo. Materials and Methods: GW was co-dissolved with poly(lactic-co-glycolic acid) (PLGA, 10%, w/v) in dichloromethane and PLGA solution was added to poly(vinyl alcohol) solution (8%, w/v). The solution was added to distilled water, and stirring was continued to obtain spheres that were then sieved to obtain a size of 20-32 µm. Poly(ethylene glycol) diacrylate (PEGDA) spheres containing the PLG microspheres were produced using a continuous-flow emulsion and photocrosslinking technique. The continuous phase consisted of mineral oil with 0.5% Span 80, while the dispersed phase was a solution of PLGA spheres, 30% PEGDA and 8% Lithium phenyl-2,4,6-trimethylbenzoylphosphinate in a functionalized collagen (Acrylate-PEG-Collagen) solution. ELISA and qRT-PCR for osteogenic markers were used to assess osteogenesis. Results and Discussion: An osteogenic microsphere was developed to coadminister hMatrix and GW to enhance hMSC retainment and promote osteorepair in vivo. Initial feasibility was demonstrated using collagen type I since this is the major component of hMatrix. We have functionalized collagen type I with photo-cross-linkable groups[4] to enable their incorporation into PEGDA microspheres. We also loaded GW into PLGA nanoparticles which were encapsulated in the PEGDA spheres and quantified the drug release profile. Specific gene and protein detection by qRT-PCR and osteoprotegerin assay of the adhered hMSCs revealed that GW induced osteogenesis. Conclusions: Conventional hMSC therapies have had limited success in bone regeneration. Autologous bone grafting is the gold standard to regenerate critical sized bone defects. Conversely, this procedure is associated with donor site morbidity and a limited volume of material available. The use of bone morphogenetic proteins have also been used, however, their use is associated with ectopic bone growth, paralysis, and inflammation[5]-[9]. Our composite microspheres incorporate biopolymers such as hMatrix to provide a favorable microenvironment and signal to cells to enhance their performance in vivo. We have developed a method to create microspheres for bone regeneration that provides both chemical and bioactive cues to promote new bone growth for critical sized bone defects.

Heterotopic bone formation in the musculus latissimus dorsi of sheep using β-tricalcium phosphate scaffolds: evaluation of an extended prefabrication time on bone formation and matrix degeneration

We previously generated viable heterotopic bone in living animals and found that 3 months of intrinsic vascularization improved bone formation and matrix degeneration. In this study, we varied the pre-vascularization time to determine its effects on the kinetics of bone formation and ceramic degradation. Two 25-mm-long cylindrical β-tricalcium phosphate scaffolds were filled intraoperatively with autogenous iliac crest bone marrow and implanted in the latissimus dorsi muscle in six sheep. To examine the effect of axial perfusion, one scaffold was surgically implanted with (group C) or without (group D) a central vascular bundle. All animals were sacrificed 6 months postoperatively and histomorphometric measurements were compared to previous results. All implanted scaffolds exhibited ectopic bone growth. However, bone growth was not significantly different between the 3-month (group A, 0.191±0.097 vs. group C, 0.237±0.075; P=0.345) and 6-month (group B, 0.303±0.105 vs. group D, 0.365±0.258; P=0.549) pre-vascularization durations, regardless of vessel supply; early differences between surgically and extrinsically vascularized constructs disappeared after 6 months. Here, we describe a reliable procedure for generating ectopic bone in vivo. A 3-month pre-vascularization duration appears sufficient and ceramic degradation proceeds in accordance with bone generation, supporting the hypothesis of cell-mediated resorption.

Biodegradable chitosan nanoparticle coatings on titanium for the delivery of BMP-2.

A simple method for the functionalization of a common implant material (Ti6Al4V) with biodegradable, drug loaded chitosan-tripolyphosphate (CS-TPP) nanoparticles is developed in order to enhance the osseointegration of endoprostheses after revision operations. The chitosan used has a tailored degree of acetylation which allows for a fast biodegradation by lysozyme. The degradability of chitosan is proven via viscometry. Characteristics and degradation of nanoparticles formed with TPP are analyzed using dynamic light scattering. The particle degradation via lysozyme displays a decrease in particle diameter of 40% after 4 days. Drug loading and release is investigated for the nanoparticles with bone morphogenetic protein 2 (BMP-2), using ELISA and the BRE luciferase test for quantification and bioactivity evaluation. Furthermore, nanoparticle coatings on titanium substrates are created via spray-coating and analyzed by ellipsometry, scanning electron microscopy and X-ray photoelectron spectroscopy. Drug loaded nanoparticle coatings with biologically active BMP-2 are obtained in vitro within this work. Additionally, an in vivo study in mice indicates the dose dependent induction of ectopic bone growth through CS-TPP-BMP-2 nanoparticles. These results show that biodegradable CS-TPP coatings can be utilized to present biologically active BMP-2 on common implant materials like Ti6Al4V.

Bone morphogenetic protein 2–mediated mandible reconstruction successfully heals bony defects but inhibits concurrent inferior alveolar nerve grafting: a rabbit experimental model.

Bone morphogenetic protein 2 (BMP-2) has been used to reconstruct mandibular defects. An elegant addition to this reconstruction method would be incorporation of a nerve graft wrapped in a BMP-2 carrier to reconstitute the inferior alveolar nerve (IAN) and restore sensation to the lower face. We developed a rabbit model to determine the effect BMP-2 has on nerve regeneration following neurorrhaphy. An inferior border mandibulectomy was created in 16 adult New Zealand white rabbits. The IAN was protected, divided, and repaired with either primary neurorrhaphy or reverse autografts. Bone defects were treated with no treatment controls (n = 2), absorbable collagen sponge (ACS) (vehicle controls) (n = 7), and ACS soaked in BMP-2 (treatment group) (n = 7). Animals underwent computed tomography (CT) 2 days and 6 weeks postoperatively. The percent bone defect healing was calculated using Amira 3D imaging software. At 6 weeks, IANs were harvested mesial to the reconstruction and were evaluated with toluidine blue histology to identify myelinated axons. Reconstructed mandible segments were evaluated with micro-CT and hematoxylin-eosin histology. Bone morphogenetic protein 2-treated animals demonstrated significantly more bone healing than did the ACS and empty defect groups (82%, 38%, 44%, respectively; P < 0.01). One hundred percent of ACS-treated nerves (n = 4) demonstrated axon regrowth, whereas only 25% of BMP-2-treated nerves (n = 4) did. Micro-CT and histology showed BMP-2 caused bone growth around the IAN, but regenerated bone infiltrated the repair site and created a physical barrier to axon growth. Bone morphogenetic protein 2 can successfully heal bone defects in the rabbit mandible, but ectopic bone growth can inhibit IAN recovery after repair. Level of Evidence: Not gradable.

Reduction of ectopic bone growth in critically-sized rat mandible defects by delivery of rhBMP-2 from kerateine biomaterials

Absorbable collagen sponges (ACS) are used clinically as carriers of recombinant human bone morphogenetic protein 2 (rhBMP-2) to promote bone regeneration. ACS exhibit ectopic bone growth due to delivery of supraphysiological levels of rhBMP-2, which is particularly problematic in craniofacial bone injuries for both functional and esthetic reasons. We hypothesized that hydrogels from the reduced form of keratin proteins (kerateine) would serve as a suitable alternative to ACS carriers of rhBMP-2. The rationale for this hypothesis is that keratin biomaterials degrade slowly in vivo, have modifiable material properties, and have demonstrated capacity to deliver therapeutic agents. We investigated kerateine hydrogels and freeze-dried scaffolds as rhBMP-2 carriers in a critically-sized rat mandibular defect model. ACS, kerateine hydrogels, and kerateine scaffolds loaded with rhBMP-2 achieved bridging in animals by 8 weeks as indicated by micro-computed tomography. Kerateine scaffolds achieved statistically increased bone mineral density compared to ACS and kerateine hydrogels, with levels reaching those of native bone. Importantly, both kerateine hydrogels and kerateine scaffolds had significantly less ectopic bone growth than ACS sponges at both 8 and 16 weeks post-operatively. These studies demonstrate the suitability of keratins as rhBMP-2 carriers due to equal regenerative capacity with reduced ectopic growth compared to ACS.

Transforaminal Lumbar Interbody Fusion With rhBMP-2 in Spinal Deformity, Spondylolisthesis, and Degenerative Disease—Part 2

Retrospective review of prospectively collected data. Without industry funding, the study evaluated short- and long-term complications related to off-label bone morphogenetic protein (BMP) used with transforaminal lumbar interbody fusion (TLIF) from a large consecutive series. Complications and results were analyzed by BMP dose, fusion length, and primary versus revision surgery. Based on the results, surgical technique and BMP dose recommendations were proposed. Off-label use of BMP in TLIF, although common, has only been studied in small series and case reports using various techniques, cage types, and doses of BMP. Several of these studies have reported minimal complications. Others report problems related to BMP, which has led to questions regarding current widespread use of TLIF with BMP. TLIF with rhBMP-2 was performed at 872 discs in 509 consecutive adults who underwent open posterior instrumented fusion and had minimum 2-year follow-up; diagnoses included degenerative disease (179), spondylolisthesis (207), deformity (123). Patient age averaged 61 years: 12% were smokers and 41% had revision surgery. TLIF was performed at 1.7 levels: single level: 229, 2 levels: 201, 3 levels: 74, 4 levels: 5. Local autograft was used for backfill around and behind each rectangular cage. Varying doses of interbody BMP were used at an average 7.3 mg per disc (range: 2-12 mg per disc). At 5 years average follow-up, 8 patients developed pseudoarthrosis at levels of TLIF (8 of 872 discs, 0.92%). Seroma (0.4%) and ectopic bone growth (0.6%) were too infrequent to be associated with a particular BMP dose. Deep infection was 2.6% overall (1.7% of the degenerative group). Symptomatic osteolysis or cage subsidence did not occur. Significant long-term improvement was noted in clinical and functional outcomes compared with preoperation. Five-year follow-up after TLIF with BMP, independent of industry, confirms effective arthrodesis in short and long fusions, both primary and revision. Most complications occurred in deformity patients. BMP-related complications (seroma, ectopic bone) were rare. 3.

Induction of Apoptosis in the Bone Marrow Promotes Regenerative Actions of Parathyroid Hormone (PTH) in Bone.

PTH has bone regenerative properties via mechanisms that remain elusive. This study aimed to determine the impact of marrow apoptosis on PTH anabolic actions. Studies were in conformance with FASEB animal use principles. Adult mice were treated with pro‐apoptotic etoposide (ETOP) or vehicle (VEH), daily 5d then 3d/wk for 6wks. PTH or VEH was administered daily for: VEH, PTH, ETOP and PTH+ETOP and skeletal phenotyping, flow cytometry, and serum bone markers analyzed. MicroCT and histomorphometry of tibiae revealed ETOP decreased and PTH increased bone volume and area, the greatest increase with PTH + ETOP. ETOP (5d) increased early marrow apoptotic cells (AnnexinV+PI‐) as well as MER+CD11b+F4/80+ and MER+CD68+F4/80+ myeloid cells. At 6wks, CD68+, Gr1+CD11b+F4/80+, CD45+and Lin−CD29+sca1+ cells were increased in ETOP w/ or w/o PTH. Serum TRAcP5b was increased in ETOP and to a greater extent with PTH. P1NP levels were increased in PTH and PTH+ETOP. PTH+ETOP mice had reduced osteoclasts per tibial bone vs. VEH or ETOP. In another model, luciferase+bone marrow stromal cells were implanted to form ectopic ossicles and bone growth tracked via bioluminescence. ETOP mice had increased luciferase vs. VEH, and ETOP augmented the PTH increase. These data suggest that induction of apoptosis provides a conducive environment for PTH pro‐regenerative actions, effects which may be due to increased macrophage efferocytosis.

Osteoinductivity Assessment of BMP-2 Loaded Composite Chitosan-Nano-Hydroxyapatite Scaffolds in a Rat Muscle Pouch.

The objective of this study was to evaluate the osteoinductivity of composite chitosan-nano-hydroxyapatite scaffolds in a rat muscle pouch model. Previous in vitro characterization demonstrated the ability of the scaffolds to promote bone regeneration and as a carrier for local delivery of BMP-2. Composite microspheres were prepared using a co-precipitation method, and scaffolds were fabricated using an acid wash to adhere beads together. To determine the in vivo osteoinductivity of the scaffolds, the following groups (n = 6) were implanted into muscle pouches created in the latissimus dorsi of Sprague Dawley rats: (A) lyophilized scaffolds without rhBMP-2, (B) lyophilized scaffolds with rhBMP-2, (C) non-lyophilized scaffolds with rhBMP-2, and (D) absorbable collagen sponge with rhBMP-2 (control). Groups B, C, and D were loaded with 4 mL of a 9.0 μg/mL solution of rhBMP-2 for 48 h. The rats were sacrificed after one month and samples were analyzed for amount of residual implant material, new bone, and osteoid. Although the experimental groups displayed minimal degradation after one month, all of the scaffolds contained small amounts of woven bone and considerable amounts of osteoid. Approximately thirty percent of the open space available for tissue ingrowth in the scaffolds contained new bone or osteoid in the process of mineralization. The ability of the composite scaffolds (with and without BMP-2) to promote ectopic bone growth in vivo was demonstrated.

Clarifying the Structure and Bone Mineral Content of Heterotopic Ossification

Heterotopic ossification (HO) has been reported as a pathologic process characterized by ectopic bone growth in muscle and/or periarticular regions. Previous reports have speculated that HO manifests as cancellous bone, cortical bone, or woven bone. Confusion regarding HO bone morphology has resulted from radiographic assessments and light microscopy, which lack the resolution required for accurately determining advanced bone architecture. Therefore, a more thorough histologic assessment using scanning electron microscopy (SEM) and backscatter electron (BSE) imaging was needed to improve HO characterization. HO samples were collected from five adult trauma patients after surgical resection and examined with radiography, BSE, and histologic stains. BSE data demonstrated that HO was composed of a heterogeneous mixture of cortical and cancellous bone with distinct regions of fibrocartilage. Bone mineralization levels varied on a patient-specific basis, with the highest percentage of hypermineralization occurring in the oldest patient. BSE and histologic stains also indicated HO remodeling continued even after 3 y from injury to resection, as evident by osteoclastic resorption and osteoid deposition. BSE provided a more accurate understanding of HO bone mineralization and structure which may lead to improved surgical planning and treatment strategies for prevention of HO recurrence after resection.

Prefabrication of vascularized bioartificial bone grafts in vivo for segmental mandibular reconstruction: experimental pilot study in sheep and first clinical application

The key elements for bioartificial bone formation in 3D matrices are large numbers of osteogenic cells and supplies of oxygen and nutrition. Vascularization becomes more important with the increasing size and complexity of seeded scaffolds required for clinical application in reconstructive craniomaxillofacial surgery. Prefabrication of vascularized bioartificial bone grafts in vivo might be an alternative to in vitro tissue engineering techniques. Two cylindrical beta-TCP-scaffolds (25 mm long) were intraoperatively filled with autogenous bone marrow from the iliac crest for cell loading and implanted into the latissimus dorsi muscle in 12 sheep. To determine the effect of axial perfusion, one scaffold in each sheep was surgically supplied with a central vascular bundle. Sheep were killed 3 months after surgery. Histomorphometric analysis showed autogenous bone marrow from the iliac crest was an effective source of osteogenic cells and growth factors, inducing considerable ectopic bone growth in all implanted scaffolds. Bone growth, ceramic resorption and angiogenesis increased significantly with axial perfusion. The results encourage the application of prefabricated bioartificial bone for segmental mandibular reconstruction in man. In clinical practice, vascularized bioartificial bone grafts could change the principles of bone transplantation with minimal donor site morbidity and no shape or volume limitations.