Keratin Scaffold Formulation Impacts rhBMP-2 Biodistribution and Bone Regeneration in a Rat Femur Defect Model

Abstract

Background: Treatment for nonunion in long bones remains a clinical need. Collagen sponges loaded with recombinant human bone morphogenetic protein 2 (rhBMP-2) are potential grafts but have limited FDA-approved applications due to safety concerns associated with rapid collagen resorption and burst release of rhBMP-2 in vivo. This work investigates keratin proteins obtained from human hair as a potential rhBMP-2 biomaterial carrier. Keratins are an appealing carrier because the extent of disulfide crosslinking can be modulated by the form of keratin present, thus allowing control over the rate of scaffold degradation.

Methods: The two forms of keratin used to formulate carriers were reductively extracted keratin called kerateine (KTN) that can form disulfide crosslinks and oxidatively extracted keratin called keratose (KOS) that does not form disulfide crosslinks. Five formulations of freeze-dried keratin scaffolds containing variable amounts of KOS and KTN were fabricated and implanted into a critically-sized rat femur defect model.

Results: A 50:50 KOS:KTN formulation with rhBMP-2 showed the same level of bone bridging, bone mineral density, and bone volume as collagen with rhBMP-2 by 8 weeks as determined by μ-CT. Scaffolds with the 50:50 KOS:KTN or 100% KTN showed approximately fourfold higher retention of fluorescently-labeled rhBMP-2 at the implant site 1, 3, or 7 days post-implant compared to collagen or 100% KOS scaffolds. The increased retention with 50:50 KOS:KTN or 100% KTN correlated with decreased levels of fluorescent rhBMP-2 in distal organs.

Conclusions: Keratin scaffolds could provide comparable levels of bone regeneration as collagen carriers with improved safety profiles suitable for use in long bone nonunions.

Keywords: Biomaterials; Fracture; Natural materials; Nonunion; Recombinant human bone morphogenetic protein.

Fig. 1

Lateral view μ-CT renderings of left femurs 4 weeks after implantation (or no treatment/empty control)

Fig. 2

Lateral view μ-CT renderings of left femurs 8 weeks after implantation (or no treatment/empty control)

Fig. 3

Analysis of μ-CT renderings of left femurs at 4 and 8 weeks after implantation. A Number of rats that showed bone bridging for each treatment group. B Bone volume for each treatment group. C Bone mineral density for each treatment group. N = 2 for empty, N = 3 for KOS-only, N = 2 for KTN-only, and these groups are combined as near replicates shown as Combined Controls. The results for these groups (empty, KOS-only, KTN-only) individually are shown as inserts for (B) and (C). None of the Combined Controls showed bridging. N = 6 for all other treatment groups. Error bars are standard error of the mean. Statistical analyses were conducted for the 4-week and 8-week time points for (B) and (C). Groups that share a letter were not found to be statistically different (P > 0.05) while groups that do not share a letter were found to be statistically different (P < 0.05). There were also no differences within groups between the 4 and 8 week time points except for BMD for the 50:50 and KTN groups, as indicated by * (P < 0.05)

Fig. 4

Mechanical properties of left femurs at time of explantation (8 weeks after implantation) as determined by 1 mm/min with 1000 N load cell. A Max load at fracture. B Elastic modulus as determined from engineering stress and engineering strain. N = 5 all groups except 70:30 KOS:KTN where the sample broke in the Instron prior to testing. None of the groups had all 6 femurs from experimental animals subjected to mechanical testing because one sample was taken for histology. Max load and modulus could not be determined for Combined Controls because the tissue at the defect site was too weak to load into the mechanical testing apparatus. Error bars denote standard error of the mean

Fig. 5

A In vitro release of AF488-rhBMP-2 from keratin hydrogels showing similar release for each of the formulations. B The average percent retention of DL800-rhBMP-2 at 1, 3, and 7 days. C Images of DL800-rhBMP-2 retention at femur defect site for three keratin formulations and for collagen at 1, 3, or 7 days post-implant. N = 4 for Day 1 and Day 3, N = 5 for Day 7 and error bars represent standard error of the mean

Fig. 6

Summary of DL800-rhBMP-2 biodistribution as indicated by the number of fluorescent events in A spleen, B liver, C kidneys, D lungs at Day 1 , Day 3 , Day 7 , of the total () of all three days. N = 4 for Day 1 and Day 3, N = 5 for Day 7 and therefore N = 13 for the Total for both spleen and liver. For kidneys and lungs, both the left and right organs were observed, so N = 8 for Day 1 and Day 3 and N = 10 for Day 7. E ndicates the total of all fluorescent events for all tissues. N = 16 (4 for spleen, 4 for liver, 8 for kidney, 8 for lung) at Day 1 and Day 3 and N = 20 (5 for spleen, 5 for liver, 10 for kidney, 10 for lung) at Day 7

Fig. 7

Possible mechanism of action for keratin (or collagen) carriers of rhBMP-2, showing impacts of biomaterial degradation and rhBMP-2 retention. Initially (top) all formulations have equal amounts of the material carrier (collagen, KOS, KOS:KTN mixtures, or KTN) and rhBMP-2. After time (bottom panel), KOS has rapid loss of the biomaterial at the implant site (white color) with time due to the lack of disulfide crosslinking. This loss of the material carrier leads to loss of rhBMP-2 from the implant that promotes rapid bone formation but with both local (ectopic growth at implant site) and systemic (rhBMP-2 in distal organ) effects. Some aspects of this (rapid degradation and rhBMP-2 in distal organs) is also true for collagen carriers. After time, KTN (bottom, right) has greater retention of rhBMP-2 at the implant site. However, the slow degradation of KTN may impede bridging because KTN remains at the implant site. The KTN:KOS mixtures (bottom, center) may provide some benefits of both systems. The degradation of KOS may allow cellular infiltration and subsequent bone bridging with rhBMP-2 release locally. However, the presence of KTN may aid in retention of more rhBMP-2, lessening local and especially systemic effects