Histological Comparison of Porcine Small Intestine Submucosa and Bovine Type-I Collagen Conduit for Nerve Repair in a Rat Model.

Purpose: After nerve injury, macrophages and Schwann cells remove axon and myelin debris. We hypothesized that nerves repaired with different conduit materials will result in varying levels of these cell populations, which impacts Wallerian degeneration and axonal regeneration. Methods: We performed a unilateral sciatic nerve transection in 18 rats. The nerves were repaired with small intestine submucosa (SIS, n = 9) or isolated type-I collagen (CLC, n = 9) conduits. Rats were monitored for 4 weeks. Histology samples were obtained from the proximal nerve, mid-implant, and distal nerve regions. Samples were stained for total macrophages, M2 macrophages, foamy phagocytes, Schwann cells, vascular components, axon components, and collagen density. Results: Distal nerve analyses showed higher populations of total macrophages and M2 macrophages in SIS-repaired nerves and higher density of foamy phagocytes in CLC-repaired nerves. Proximal nerve, mid-implant, and distal nerve analyses showed higher Schwann cell and vascular component densities in SIS-repaired nerves. Axon density was higher in the mid-implant region of SIS-repaired nerves. Collagen staining in the mid-implant was scant, but less collagen density was observed in SIS-repaired versus CLC-repaired nerves. Conclusions: In the distal nerve, the following were observed: (1) lower total macrophages in CLC-repaired nerves, suggesting lower overall inflammation versus SIS-repaired nerves; (2) higher M2 macrophages in SIS-repaired versus CLC-repaired nerves, a driving factor for higher total macrophages and indicative of an inflammation resolution response in SIS-repaired nerves; and (3) a lower foamy phagocyte density in SIS-repaired nerves, suggesting earlier resolution of Wallerian degeneration versus CLC-repaired nerves. In the proximal nerve, mid-implant, and distal nerve, higher Schwann cell and vascular component densities were noted in SIS-repaired nerves. In the mid-implant, a higher axon component density and a lower collagen density of the SIS-repaired nerves versus CLC-repaired nerves were noted. These results indicate more robust nerve regeneration with less collagen deposition. Clinical relevance: This in vivo study evaluated two common conduit materials that are used in peripheral nerve repair. Clinical outcomes of nerves repaired with conduits may be impacted by the response to different conduit materials. These nerve repair responses include Wallerian degeneration, nerve regeneration, and nerve scarring. This study evaluated Wallerian degeneration using total macrophages, M2 macrophages, and foamy phagocytes. Nerve regeneration was evaluated using Schwann cells and axons. Nerve scarring was evaluated using vascular and collagen density.

Characterization of Nerve Damage After an Injury to the Adjacent Soft Tissue: A Pilot Animal Study.

Traumatic injuries may result in the formation of soft tissue adhesions between peripheral nerves and surrounding soft tissue. These soft tissue adhesions lead to compression and ischemic stress within fascicles due to nonpliability of adhered scar tissue, and nerve tension due to loss of nerve gliding from scar tethering. These changes in the soft tissue bed surrounding the nerve may result in axon degeneration and neuroma-in-continuity. Preclinical models that simulate clinically relevant levels of scar in the nerve environment may be impactful to the development of surgical techniques and treatments to prevent adhesions. This study presents the results of a rodent model with an induced indirect nerve injury by (1) thermal insult to the soft tissue bed surrounding the nerve and (2) air-drying the surrounding soft tissue bed of the nerve. Our findings suggest that inducing an injury of the soft tissue bed results in increased intraneural scar and extraneural adhesions to the nerve compared to a sham procedure. Thermal induced injuries showed more macrophages and changes in nerve health compared to air-dried induced injuries. The changes in the nerves of the induced injury groups, specifically the thermal injury group, may be meaningful for evaluating treatments for nontransected nerve injuries.

A Comparative Study of Porcine Small Intestine Submucosa and Cross-Linked Bovine Type I Collagen as a Nerve Conduit.

Purpose: We compared 2 commercially available nerve conduits-the Axoguard Nerve Connector, made of porcine small intestine submucosa (SIS), and the NeuraGen Nerve Guide, made of cross-linked bovine type I collagen (Col)-using a rodent model at 4 weeks, specifically focusing on subchronic host responses to the implants. Methods: A unilateral 5-mm sciatic nerve defect was created in 18 male Lewis rats and was repaired with SIS or Col conduits. After 4 weeks, histological evaluations of morphology, collagen content, macrophage polarization, vascularization, axonal regeneration, and myelination were conducted. To achieve a blinded examination, an independent qualified pathologist evaluated the images that were stained with hematoxylin-eosin, α-smooth muscle actin, and Masson trichrome stains. Results: The results showed a dominant macrophage type 2 (M2) response in the SIS group and a dominant macrophage type 1 (M1) response in the Col group. The SIS group showed deeper implant vascularization and fibroblast ingrowth than the Col group. Collagen deposition was higher within the lumen of the Col group than the SIS group. All Col conduits were surrounded by a colocalized staining of Masson trichrome and α-smooth muscle actin, forming a capsule-like structure. Conclusion: Distinctive histological features were identified for each conduit at the cellular level. The SIS conduits had a significantly higher number of host macrophages expressing M2 surface marker CD163, and the Col conduits showed a predominance of host macrophages expressing the M1 surface marker CD80. Data suggest that promoting the M2 response for tissue engineering and regenerative medicine is associated with a remodeling response. In addition, an independent analysis revealed an encapsulation-like appearance around all Col conduits, which is similar to what is seen in breast implant capsules. Clinical relevance: The biomaterial choice for conduit material can play an important role in the host tissue response, with the potential to impact adverse events and patient outcomes.

A comparative study of three biomaterials in an ovine bone defect model.

BACKGROUND: Polyetheretherketone (PEEK), and more recently titanium-coated PEEK, have been given serious consideration as biomaterial design choices for spinal interbody implants. Shortcomings in these materials necessitate further innovation into materials research, for example, on PEKK. Common complications such as surface delamination (as with titanium coating) and lack of bone apposition (as with PEEK) indicate the need for a new material that inherently displays preferable bone growth characteristics without sacrificing structural integrity. PURPOSE: To compare three biomaterials with respect to their osseointegrative capacity. STUDY DESIGN: Evaluate the in vivo material characteristics of three separate biomaterials in an ovine bone defect model: PEEK, titanium-coated PEEK (Ti-coated PEEK), and 3D-printed PEKK. Biomechanical, histologic, and radiographic testing was the basis for evaluation and material characterization. METHODS: Eight ovine specimens were implanted with one of each of the three types of biomaterials tested in both left and right epicondyles using a femoral bone defect model, and were sacrificed at 8 and 16 weeks. Implants were then analyzed using a push-out method, histologic staining, and various radiographic tests. Industry funding was provided for the completion of this research study, followed by an independent third party review of all relevant data for publication. RESULTS: PEKK implants demonstrated bone ingrowth, no radiographic interference, no fibrotic tissue membrane formation, significant increase in bony apposition over time, and significantly higher push-out strength compared to standard PEEK. The PEKK implant displayed bone growth characteristics comparable to Ti-coated PEEK with significant improvements in implant integrity and radiographic properties. CONCLUSION: This study found that PEKK displayed preferable characteristics when compared to PEEK and Ti-coated PEEK, and is therefore a potential alternative to their use.

Multipotent adult progenitor cell-loaded demineralized bone matrix for bone tissue engineering.

Multipotent adult progenitor cells (MAPCs) from bone marrow have been shown to be capable of forming bone, cartilage and other connective tissues. In addition, MAPCs differentiate into lineages that are different from their germ layers of origin. Previous studies showed the ability of MAPCs to improve cardiac function and control allogenic-reactive responses associated with acute graft versus host disease. In the current study, we evaluated the ability of MAPCs to produce bone matrix on demineralized bone allograft substrates. Specifically, MAPCs expressed alkaline phosphatase, produced extracellular matrix proteins and deposited calcium-containing mineral on demineralized bone matrices. Furthermore, the addition of MAPCs on demineralized bone matrix (DBM) scaffolds enhanced osteoinductivity of the carrier in a rat ectopic pouch model. These results demonstrated the potential of MAPCs as a new approach for bone repair in tissue-engineering applications.

Local antibiotic delivery with demineralized bone matrix.

A method of care for these infected nonunions is prolonged intravenous systemic antibiotic treatment and implantation of methyl methacrylate antibiotic carrier beads to delivery high local doses of antibiotics. This method requires a second surgery to remove the beads once the infection has cleared. Recent studies have investigated the use of biodegradable materials that have been impregnated with antibiotics as tools to treat bone infections. In the present study, human demineralized bone matrix (DBM) was investigated for its ability to be loaded with an antibiotic. The data presented herein demonstrates that this osteoinductive and biodegradable material can be loaded with gentamicin and release clinically relevant levels of the drug for at least 13 days in vitro. This study also demonstrates that the antibiotic loaded onto the graft has no adverse effects on the osteoinductive nature of the DBM as measured in vitro and in vivo. This bone void filler may represent a promising option for local antibiotic delivery in orthopedic applications.

Osteoconductivity and osteoinductivity of Puros(R) DBM putty.

Bone graft substitutes have been developed due to the limited supply and morbidity associated with using autogenous graft material. Allogeneic demineralized bone matrix (DBM) has been used extensively as a clinical graft material because of its inherent osteoinductive and osteoconductive properties. Differential enhancement of these properties may optimize the performance of these products for various orthopedic and craniofacial applications. Commercially available bone paste products consist of formulations that combine DBM with a carrier to facilitate handling and containment. In the present study, we present results of a comprehensive in vitro and in vivo characterization of a 100% human DBM putty product, Puros DBM Putty. Results indicate the DBM particles are completely dispersed in the putty. Data are presented showing the porosity of and cell attachment to Puros DBM Putty, thereby demonstrating the osteoconductive properties of this DBM. Puros DBM Putty was also shown to be osteoinductive in the rat ectopic pouch model. We demonstrate here for the first time that Puros DBM Putty maintains its activity to markedly stimulate or induce bone formation over the entire period of its shelf life. Taken together, these data demonstrate that the 100% human allograft derived Puros DBM Putty could be an effective bone graft substitute.

Human adipose-derived side population stem cells cultured on demineralized bone matrix for bone tissue engineering.

BACKGROUND: Tissue engineering of new bone relies on the combination and application of osteoconductive, osteoinductive, and osteogenic elements. Natural scaffolds, such as demineralized bone matrix (DBM), contain collagenous networks with growth factors such as bone morphogenetic protein-2. Stem cells from readily available sources, including discarded adipose tissue, have the propensity to differentiate into bone. The present study examines a multi-component technique consisting of a novel side population of adipose stem cells cultured on DBM for tissue engineering applications. METHODS: Adipose-derived side population stem cells were cultured on DBM for up to 14 days. Cell proliferation, alkaline phosphatase activity, extracellular matrix protein production, and calcium-containing mineral deposit formation were assayed. Ectopic bone formation in a rat model was also evaluated. RESULTS: Side population stem cells attached to and proliferated on DBM while generating markers of new bone formation. When these cell/substrate composites were implanted into an ectopic model, newly formed bone was 30% greater than that of DBM alone. CONCLUSIONS: Novel populations of adipose-derived stem cells cultured on DBM compose a system that develops new bone matrix in vitro and in vivo. This strategy provides a novel approach using naturally occurring materials for bone repair in tissue engineering applications.

Histological and radiographic evaluations of demineralized bone matrix and coralline hydroxyapatite in the rabbit tibia.

Complex fractures resulting in bone loss or impaired fracture healing remain problematic in trauma and orthopedic surgeries. Many bone graft substitutes have been developed and are commercially available. These products differ in their osteoconductive and osteoinductive properties. Differential enhancement of these properties may optimize the performance of these products for various orthopedic and craniofacial applications. The use of bone graft substitutes offers the ability to lessen the possible morbidity of the harvest site in autografts. The objective of the present study was to compare the ability of two bone graft substitutes, BioSet RT, an allograft demineralized bone matrix formulation, and ProOsteon 500R, a coralline hydroxyapatite, in a rabbit critical tibial defect model. BioSet RT and ProOsteon 500R were implanted into a unicortical proximal metaphyseal tibial defect and evaluated for new bone formation. Samples were analyzed radiographically and histologically at 1 day, 6 weeks, 12 weeks, and 24 weeks post surgery. Both materials were biocompatible and demonstrated significant bone growth and remodeling. At 12 weeks, the BioSet RT implanted sites demonstrated significantly more defect closure and bone remodeling as determined by radiographic analyses with 10 out of 14 defects being completely healed versus 1 out of 14 being completely healed in the ProOsteon 500R implanted sites. At 24 weeks, both materials demonstrated complete closure of the defect as determined histologically. There were no statistical differences in radiographic scores between the two implanted materials. However, there was an observable trend that the BioSet RT material generated higher histological and radiographic scores, although not statistically significant. This study provides evidence that both BioSet RT and ProOsteon 500R are biocompatible and able to induce new bone formation as measured in this rabbit model. In addition, this in vivo study demonstrates the ability of BioSet RT to induce new bone formation in a shorter timeframe than ProOsteon 500R.

Biomechanical and radiographic comparison of demineralized bone matrix, and a coralline hydroxyapatite in a rabbit spinal fusion model.

The use of bone grafts is an essential component in spinal fusion. Autologous bone has been shown to result in long-term stable arthrodesis between spinal motion segments. However, autograft can be associated with significant morbidity and a limited supply. Alternatives, such as allogeneic demineralized bone matrix (DBM), are a potential source and supplement to autograft bone. The current study compares the ability of a DBM product (BioSet RT) and a coralline hydroxyapatite (Pro Osteon 500R), for inducing spinal fusion in a rabbit model. BioSet RT, alone or in combination with autograft, and Pro Osteon 500R were implanted in the posterior lateral inter-transverse process region of the rabbit spine. The spines were evaluated at 18 weeks for fusion of the L4-L5 transverse processes using a total of 33 skeletally mature male rabbits; 4 naïve animals were also included in the study. Samples were evaluated radiographically, histologically, by palpation, and through mechanical strength testing. Radiographical, histological, and palpation measurements demonstrated the ability of BioSet RT to induce new bone formation and bridging fusion comparable to autograft. This material performed well alone or in combination with autograft material. Despite significantly higher biomechanical testing results, minimal bone formation and fusion was recorded for the Pro Osteon 500R-treated group. This in vivo study demonstrates the ability of BioSet RT to induce new bone formation, and there was a clear relationship between bridging bone and mechanical strength.

Immunologic analyses of bovine bone treated with a novel tissue sterilization process.

BACKGROUND: This study evaluates the efficacy of removal of xeno-antigens from bovine bone using a patented BioCleanse process for decellularization of allograft tissues for clinical implantation. BioCleanse deploys a combination of chemicals and several high pressure rinses to achieve standardized sterility assurance levels. This method produces sterile grafts without reducing allograft bone biomechanical properties and effectively removes cells, lipids, and other sources of antigenic material from human allografts for clinical use. METHODS: In this investigation, BioCleanse is evaluated for its potential in removing xenograft antigens from bovine bone grafts followed by immunologic evaluation in the subcutaneous pouch of immunocompetent rats. The alpha-galactosyl (alpha-gal) epitope with the structure Galalpha1-3Galbeta1-4GlcNAc-R constitutes a critical component of xenoantigens and its removal using BioCleanse from bovine bone was compared with tissue levels of unprocessed bone. The relative degree of antigen removal was also determined through measuring the pro-inflammatory cytokine, tumor necrosis factor (TNF)-alpha, and through the use of histologic grading of cellular infiltrates into bone. RESULTS: Compact cortical bone inhibited immune cell migration but cancellous bone demonstrated cellular increase and bone resorption in the untreated control group. The alpha-gal xenoantigen level was significantly lower in both cortical (P < 0.001) and cancellous bone (P < 0.001) compared with controls. TNF-alpha levels were significantly (P < 0.001) reduced compared with untreated controls when human acute monocytic leukemia cells were exposed to cortical or cancellous bone. CONCLUSIONS: BioCleanse effectively removed xenoantigens and inflammatory markers justifying a follow up study in primates to determine these benefits in a model that is primed with preformed xeno-antibodies responsible for hyperacute rejection in hard tissues.