Bone marrow mesenchymal stem cells with low dose bone morphogenetic protein 2 enhances scaffold-based spinal fusion in a porcine model.

High doses bone morphogenetic protein 2 (BMP-2) have resulted in a series of complications in spinal fusion. We previously established a polyelectrolyte complex (PEC) carrier system that reduces the therapeutic dose of BMP-2 in both rodent and porcine spinal fusion models. This study aimed to evaluate the safety and efficacy of the combination of bone marrow mesenchymal stem cells (BMSCs) and low dose BMP-2 delivered by PEC for bone regeneration in a porcine model of anterior lumbar interbody spinal fusion (ALIF) application. Six Yorkshire pigs underwent a tri-segmental (L2/L3; L3/L4; L4/L5) ALIF in four groups, namely: (a) BMSCs + 25 µg BMP-2/PEC (n = 9), (b) 25 µg BMP-2/PEC (n = 3), (c) BMSCs (n = 3), and (d) 50 µg BMP-2/absorbable collagen sponge (n = 3). Fusion outcomes were evaluated by radiography, biomechanical testing, and histological analysis after 12 weeks. Mean radiographic scores at 12 weeks were 2.7, 2.0, 1.0, and 1.0 for Groups 1 to 4, respectively. µ-CT scanning, biomechanical evaluation, and histological analysis demonstrated solid fusion and successful bone regeneration in Group 1. In contrast, Group 2 showed inferior quality and slow rate of fusion, and Groups 3 and 4 failed to fuse any of the interbody spaces. There was no obvious evidence of seroma formation, implant rejection, or any other complications in all groups. The results suggest that the combination of BMSCs and low dose BMP-2/PEC could further lower down the effective dose of the BMP-2 and be used as a bone graft substitute in the large animal ALIF model.

Dose-dependent Nerve Inflammatory Response to rhBMP-2 in a Rodent Spinal Nerve Model.

STUDY DESIGN: We developed a spinal nerve root wrapping rodent model to evaluate the relationship between recombinant human bone morphogenetic protein 2 (rhBMP-2) dosage and the degree of inflammation. OBJECTIVE: To investigate the direct effects of recombinant human bone morphogenetic protein 2 (rhBMP-2) dosage and the degree of inflammation in rodent spinal nerve roots. SUMMARY OF BACKGROUND DATA: rhBMP-2 is commonly used in clinical practice to augment spinal fusion. However, complications such as postoperative leg pain, and a higher rate of postoperative neurologic deficits have been reported. These may be attributable to the exposure of adjacent nerve roots to high doses of rhBMP-2. METHODS: Eighteen rats were randomized into three groups as follows: Group 1: absorbable collagen sponge (ACS) + 10 µg rhBMP-2, Group 2: ACS + 1 µg rhBMP-2, and Group 3 ACS with 20 µL saline. The ACS containing rhBMP-2 or saline were then wrapped around the L5 nerve root and secured loosely with nonabsorbable sutures. At 1-week postoperation, the rats were sacrificed, and the L5 nerve root and dorsal root ganglion harvested for reverse transcription polymerase chain reaction (RT-PCR), histology and immunohistochemical staining. RESULTS: In our study, 10 µg rhBMP-2 induced a 10-fold increase in seroma compared with 1 µg group. Using RT-PCR, macrophage markers MIP3-α, and CD-68 were upregulated by 8- and 2-fold respectively in comparison with the saline group. Haematoxylin and eosin (H&E) images demonstrated disruption of nerve structures in the high dose 10 µg rhBMP-2, but not at 1 µg rhBMP-2 and with saline. CONCLUSION: High doses of rhBMP-2 induced neuroinflammation in a dose dependent manner, resulting in higher seroma volume, macrophage marker gene expressions, and higher proportions of immunohistochemically stained TNF-alpha and more macrophage infiltration. LEVEL OF EVIDENCE: 2.

Bone marrow-derived mesenchymal stem cells assembled with low-dose BMP-2 in a three-dimensional hybrid construct enhances posterolateral spinal fusion in syngeneic rats.

BACKGROUND CONTEXT: The combination of potent osteoinductive growth factor, functional osteoblastic cells, and osteoconductive materials to induce bone formation is a well-established concept in bone tissue engineering. However, supraphysiological dose of growth factor, such as recombinant human bone morphogenetic protein 2 (rhBMP-2), which is necessary in contemporary clinical application, have been reported to result in severe side effects. PURPOSE: We hypothesize that the synergistic osteoinductive capacity of low-dose bone morphogenetic protein 2 (BMP-2) combined with undifferentiated bone marrow-derived stromal cells (BMSCs) is comparable to that of osteogenically differentiated BMSCs when used in a rodent model of posterolateral spinal fusion. STUDY DESIGN/SETTING: A prospective study using a rodent model of posterolateral spinal fusion was carried out. PATIENT SAMPLE: Thirty-six syngeneic Fischer rats comprised the patient sample. METHODS: Six groups of implants were evaluated as follows (n=6): (1) 10 µg BMP-2 with undifferentiated BMSCs; (2) 10 µg BMP-2 with osteogenic-differentiated BMSCs; (3) 2.5 µg BMP-2 with undifferentiated BMSCs; (4) 2.5 µg BMP-2 with osteogenic-differentiated BMSCs; (5) 0.5 µg BMP-2 with undifferentiated BMSCs; and (6) 0.5 µg BMP-2 with osteogenic-differentiated BMSCs. Optimal in vitro osteogenic differentiation of BMSCs was determined by quantitative real-time polymerase chain reaction (qRT-PCR) gene analysis whereas in vivo bone formation capacity was evaluated by manual palpation, micro-computed tomography, and histology. RESULTS: Rat BMSCs cultured in fibrin matrix that was loaded into the pores of medical-grade poly epsilon caprolactone tricalcium phosphate scaffolds differentiated toward osteogenic lineage by expressing osterix, runt-related transcription factor 2, and osteocalcium mRNA when supplemented with dexamethasone, ascorbic acid, and ß-glycerophosphate. Whereas qRT-PCR revealed optimal increase in osteogenic genes expression after 7 days of in vitro culture, in vivo transplantation study showed that pre-differentiation of BMSCs before transplantation failed to promote posterolateral spinal fusion when co-delivered with low-dose BMP-2 (1/6 or 17% fusion rate). In contrast, combined delivery of undifferentiated BMSCs with low-dose BMP-2 (2.5 µg) demonstrated significantly higher fusion rate (4/6 or 67%) as well as significantly increased volume of new bone formation (p<.05). CONCLUSION: In summary, this study supports the combination of undifferentiated BMSCs and low-dose rhBMP-2 for bone tissue engineering construct.

Polyelectrolyte Complex Carrier Enhances Therapeutic Efficiency and Safety Profile of Bone Morphogenetic Protein-2 in Porcine Lumbar Interbody Fusion Model.

STUDY DESIGN: Porcine lumbar interbody fusion model. OBJECTIVE: This study evaluates the effect of polyelectrolyte complex (PEC) carrier in enhancing the therapeutic efficiency and safety profile of bone morphogenetic protein-2 (BMP-2) in a large animal model. SUMMARY OF BACKGROUND DATA: Extremely large amounts of BMP-2 are administered to achieve consistent spinal fusion, which has led to complications. Heparin-modified PEC carrying reduced BMP-2 doses of 0.5 µg was demonstrated to achieve consistent spinal fusion with reduction of complications in rodent model. The purpose of this study was to evaluate whether PEC could improve the therapeutic efficiency of BMP-2 in porcine model. METHODS: Three-segment (L3-L6) anterior lumbar interbody fusions with instrumentation were performed on 6 pigs using 3 different doses of BMP-2, namely, (1) 50 µg, (2) 150 µg, and (3) 300 µg. The BMP-2 was delivered using heparin-modified alginate microbeads loaded into biodegradable cage. Fusion performance was evaluated after 3 months. RESULTS: Manual palpation and micro-computed tomography showed consistent fusion in all experimental groups. Heterotopic bone formation beyond the cage implant area was more evident in group 2 and group 3 than in group 1. Similarly, superior bone microstructure was observed in the new bone with the lowered BMP-2 dose. Biomechanical evaluation revealed enhanced stiffness of the operated segments compared with nonoperated segments (P < 0.05). Mechanical stability was maintained despite dose reduction of BMP-2. Although the mineral apposition rate was higher in group 3, unsatisfactory bony microstructure with decreased trabecular number was observed in group 3 compared with group 1. CONCLUSION: PEC carrying low doses of BMP-2 achieved consistent interbody fusion. We observed dose-related reduction in heterotopic ossification without compromising the stability of the fused segments. PEC carrier reduces the efficacious doses of BMP-2. This could enhance the safety profile of BMP-2 and reduce dose- and carrier-related complications. LEVEL OF EVIDENCE: N/A.

Novel Protamine-Based Polyelectrolyte Carrier Enhances Low-Dose rhBMP-2 in Posterolateral Spinal Fusion.

STUDY DESIGN: A rodent posterolateral spinal fusion model. OBJECTIVE: This study evaluated a protamine-based polyelectrolyte complex (PEC) developed to use heparin in enhancing the biological activity of low-dose recombinant human bone morphogenetic protein-2 (rhBMP-2) in spinal fusion. SUMMARY OF BACKGROUND DATA: rhBMP-2 is commonly regarded as the most potent bone-inducing molecule. However, poor pharmacokinetics and short in vivo half-life means that large amounts of the bioactive growth factor are required for consistent clinical outcomes. This has been associated with a number of adverse tissue reactions including seroma and heterotopic ossification. Glycosaminoglycans including heparin are known to stabilize rhBMP-2 bioactivity. Previous studies with poly-L-lysine (PLL) and heparin-based PEC carriers amplified the therapeutic efficacy of low-dose BMP-2. However, questions remained on the eventual clinical applicability of relatively cytotoxic PLL. In the present study, a protamine-based PEC carrier was designed to further enhance the safety and efficacy of BMP-2 by delivering lower dose within the therapeutic window. METHODS: A polyelectrolyte shell was deposited on the surface of alginate microbead templates using the polycation (protamine)/polyanion (heparin) layer-by-layer polyelectrolyte self-assembly protocol. rhBMP-2 was loaded onto the outermost layer via heparin affinity binding. Loading and release of rhBMP-2 were evaluated in vitro. The bone-inductive ability of 20-fold reduction of rhBMP-2 with the different carrier vehicle was evaluated using a posterolateral spinal fusion model in rats. RESULTS: In vitro uptake and release analysis, protamine-based PEC showed higher uptake and significantly enhanced control release than PLL-based PEC (P < 0.05). In vivo implantation with protamine-based and PLL-based PEC showed better fusion performances than absorbable collagen sponge-delivered same dose of rhBMP-2, and negative control group through manual palpation, micro-computed tomography, and histological analyses. CONCLUSION: Solid posterolateral spinal fusion was achieved with 20-fold reduction of rhBMP-2 when delivered using protamine-based PEC carrier in the rat posterolateral spinal fusion model. LEVEL OF EVIDENCE: N/A.

Preclinical evaluation of strontium-containing bioactive bone cement.

Strontium (Sr) has become more attractive for orthopaedic applications as they can simultaneously stimulate bone formation and prevent bone loss. A Sr-containing bioactive bone cement (Sr-BC) has been designed to fix osteoporotic bone fracture. Sr is a trace element, so the safety of containing Sr is concerned when Sr-BC is implanted in human body. The preclinical assessment of biocompatibility of Sr-BC was conducted according to ISO 10993 standards. MTT assay showed that this bioactive bone cement was non-toxic to mouse fibroblasts, and it met the basic requirement for the orthopaedic implant. The three independent genetic toxicity studies including Ames, chromosome aberration and bone marrow micronucleus assays demonstrated absence of genotoxic components in Sr-BC, which reassured the safety concerns of this novel bone cement. The muscle implantation results in present study were also encouraging. The acute inflammation around the cement was observed at 1 week post-implantation; however, no significant difference was observed between control and Sr-BC groups. These responses may be attributed to the presence of the foreign body, but the tissue healed after 12 weeks implantation. In summary, the above preclinical results provide additional assurance for the safety of this implant. Sr-BC can be used as a potential alternative to the traditional bone cement.

Minimizing the severity of rhBMP-2-induced inflammation and heterotopic ossification with a polyelectrolyte carrier incorporating heparin on microbead templates.

STUDY DESIGN: A rodent model of posterior spinal fusion. OBJECTIVE: The aim of this study was to evaluate the efficacy of low-dose recombinant human bone morphogenetic protein-2 (rhBMP-2) delivered with a heparin based polylectrolyte complex (PEC) carrier in facilitating posterior spinal fusion while concurrently minimizing seroma and heterotopic ossification. SUMMARY OF BACKGROUND DATA: rhBMP-2 is being used to augment spinal fusion. However, complications such as heterotopic ossification and local soft tissue swellings have been reported. These are attributed to supraphysiological amount of rhBMP-2 and the poor modulation capacity of absorbable collagen sponge. METHODS: Forty rats were randomized into 6 groups as follows. Group I: absorbable collagen sponge without rhBMP-2 (n = 4); group II: positive control, absorbable collagen sponge + 10 µg rhBMP-2 (n = 4); group III: alginate-(poly-L-lysine)-heparin (PEC) without rhBMP-2 (n = 8); group IV: PEC + 4.5 µg rhBMP-2 (n = 8); group V: PEC + 1.5 µg rhBMP-2 (n = 8); group VI: PEC + 0.5 µg rhBMP-2 (n = 8). RESULTS: Between postoperative days 5 and 7, seroma was observed in all rhBMP-2 implanted groups irrespective of carrier and dose. However, the rate and size of seroma differed considerably. Although all animals (100%) in positive control group showed seroma, only one animal (12.5%) in group VI developed seroma at the implant site. The size of seroma in group VI was significantly smaller than that in positive control group. Micro-computed tomography evaluation revealed comparable mean fusion scores in all rhBMP-2 implanted groups. More importantly, although new bone was well contained within the cage in group VI, heterotopic ossification beyond the cage was observed in positive control group. CONCLUSION: A new carrier has demonstrated capacity to minimize seroma formation as well as heterotopic ossification associated with rhBMP-2 by reducing the efficacious dose needed for consistent fusion. The results of this study indicate that PEC alginate microbeads may represent a new opportunity to define an efficient rhBMP-2 carrier.