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Year : 2020  |  Volume : 12  |  Issue : 1  |  Page : 86-91

Healing of gap nonunion using autologous cultured osteoblasts impregnated over three-dimensional bio-degradable nanomaterial scaffold: A pilot experiment on rabbits

1 Department of Orthopaedics, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India
2 School of Biochemical Engineering, Indian Institute of Technology, Banaras Hindu University, Varanasi, Uttar Pradesh, India

Date of Submission12-Nov-2018
Date of Acceptance01-Mar-2020
Date of Web Publication26-Jun-2020

Correspondence Address:
Dr. Shivam Sinha
Department of Orthopaedics, Institute of Medical Sciences, Banaras Hindu University, Varanasi - 221 005, Uttar Pradesh
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jotr.jotr_42_18

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Background: Non union is a failure of fractured bones to unite.For successful treatment of non union the addition Of autograft at fracture site is recommended but it has its own disadvantages to overcome this recent studies have focused on bone tissue engineering. Bone tissue engineering involves the use of a combination of scaffolds with osteoblasts or osteogenic potential cells to form bone tissue, which can lead to new bone formation at the affected area when implanted in vivo. Aims and Objective: To present our results with the use of a novel concept of using nanomaterial bioscaffold impregnated with cultured osteoblast over experimentally created bone defects with critical size defect in rabbits. Materials and Methods: We studied the effect of nano-hydroxyapatite-tricalcium phosphate-gelatin-chitosan scaffold impregnated with autologous cultured osteoblast on gap created on 16 iliac crests of eight rabbits. The right side was implanted, and on the left side, the gap nonunion was left as control. Rabbits were followed up at 4, 8, 12, and 20 weeks for radiographic union of scaffold and gross and microscopic examination at the final sacrifice at 20 weeks for assessment of scaffold loosening, osteogenesis, immunological reaction, and persistence of graft. Result: Statistically significant difference was found between osteointegration of scaffold, however it persisted even at 20 weeks, but no immunogenicity was observed. Conclusion: Use of nanomaterials and chitosan can attributably improve the osteogenesis by autologous osteoblasts in gap nonunion.

Keywords: Chitosan, nano-hydroxyapatite, nonunion, osteoblast, tricalcium phosphate

How to cite this article:
Jain Y, Sinha S, Rastogi A, Srivastava PK, Kumar Y, Vivek V. Healing of gap nonunion using autologous cultured osteoblasts impregnated over three-dimensional bio-degradable nanomaterial scaffold: A pilot experiment on rabbits. J Orthop Traumatol Rehabil 2020;12:86-91

How to cite this URL:
Jain Y, Sinha S, Rastogi A, Srivastava PK, Kumar Y, Vivek V. Healing of gap nonunion using autologous cultured osteoblasts impregnated over three-dimensional bio-degradable nanomaterial scaffold: A pilot experiment on rabbits. J Orthop Traumatol Rehabil [serial online] 2020 [cited 2021 May 8];12:86-91. Available from: https://www.jotr.in/text.asp?2020/12/1/86/287716

  Introduction Top

Nonunion is a failure of the ends of fractured bones to unite. In a nonunion, the progression of fracture healing has ceased, and there is motion at the fracture site.[1] For the successful treatment of nonunion bone fracture, the addition of bone autograft at the fracture site is recommended to stabilize the bone fracture.[2] A cancellous bone autograft facilitates excellent bone formation, which can lead to bone union through its osteogenesis, osteoconduction, and osteoinduction. Autocancellous grafts have the disadvantages of being limited in amount, donor site morbidity as fracture, pain, osteomyelitis, and poor harvest in pediatric population.

Gap nonunion is a type of nonunion which acclaims a critical size defect despite adequate biology fails to unite. Thus, a critical size bony defect (CSD) is defined as the smallest intraosseous wound characterized by an absence of spontaneous healing, which would not heal by bone formation during the lifetime.[3] CSD creation is the method of choice forin vivo testing of bone repair materials.[4] Most of the nonoperative strategies for the treatment of nonunion include cast brace immobilization, low-intensity pulsed ultrasound, implanted bone growth stimulator, and bone marrow injection, which have not evidently proven their clinical efficacy.[5],[6] Use of biomaterials like bone cement, calcium sulfate, hydroxyapatite (HAP), tricalcium phosphate (TCP), bio glass etc., as bone graft substitute has been in vogue since a fair amount of time and they are still in process of evolution.

Therefore, recent studies have focused on bone tissue engineering to overcome these disadvantages.[7] Bone tissue engineering involves the use of a combination of scaffolds with osteoblasts or osteogenic potential cells to form bone tissue, which can lead to new bone formation at the affected area when implanted in vivo. In human studies, there are only a few reported clinical cases of bone tissue engineering being used to treat bone defects and nonunion fractures.[8] Current challenges include the engineering of materials that can match both the mechanical and biological context of real bone tissue matrix and support the vascularization of large tissue constructs. Scaffolds with new levels of bio functionality that attempt to recreate nanoscale topographical and bio factor cues from the extracellular environment are emerging as interesting candidate biomimetic materials. Hereby, we present our results with the use of a novel concept of using nanomaterial bioscaffold impregnated with cultured osteoblast over experimentally created bone defects with critical size defect in rabbits.

  Materials and Methods Top

An experimental study was conducted in the department of orthopedics, where customized scaffold prepared by the department of biochemical engineering was used on eight rabbits. The study was duly approved by the institutional review board regarding animal experimentation.

Preparation of scaffold

The scaffold used for osteoblast culture must have sufficient biocompatibility, biodegradability, porosity, strength, and should be easy to prepare. Nano-hydroxyapatite (NHAP)/TCP/gelatin/chitosan composites scaffolds were fabricated using freeze-dried technique. HAP nanocrystal powder was dispersed in a solvent by sonication for 30 s at 15 W (virsonic 100, Cardiner, NY, USA). After that, TCP was dissolved in the NHAP-suspended solvent (dioxane) containing gelatin and chitosan at about 40°C to make homogenous solutions with desired concentrations. A volume of 2.5 ml polymer NHAP mixture was added into a Teflon vial, sonicated again, and then transferred into a freezer at a preset temperature of −20°C to induce solid–liquid or liquid–liquid phase separation. The solidified mixture was maintained at that temperature overnight and then transferred to a freeze-drying container which was maintained at a temperature between 5°C and 10°C salt-ice bath for freeze drying. The samples were freeze dried at 0.5 mmHg for 7 days to remove solvent. The final composition of the composite foam was determined by the concentration of the polymer solution and NHAP content in mixture.

Procuring animals

Eight healthy white adult male rabbits of average 2–3 kg weight were chosen and left for 1 week before starting experiment. The rabbits were kept in a smooth-walled, stainless-steel colony cage (1 animal per cage) at an ambient temperature of 25°C and 45%–55% relative humidity with 10h: 14h, light: dark cycle and maintained under pathogen-free condition. They had free access to standard diet (pellet) and water ad libitum.

The principle of laboratory care, feeding, and sacrifice was followed as per ethics provided by INSA, Animal Welfare Division of the Ministry of Environment and Forest, Council of International Organisation of Medical Sciences (WHO/United Nations Educational, Scientific and Cultural Organization), National Institutes of Health and PHS.

Operative procedure

Surgical procedure was performed under anesthesia using ketamine and midazolam injection intramuscularly. In each rabbit, both iliac crests were shaved and disinfected with spirit and betadine [Figure 1]. The right-sided iliac crest is exposed using incision over the anterior superior iliac spine extending posteriorly over the iliac crest, and 1-cm bone defect is created using a bone cutter. The incision site was sutured and dressed, and the rabbits were transferred back to their cages and allowed to ambulate after adequate analgesia.
Figure 1: Part preparation and draping of rabbit iliac crest

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Cell isolation

The osteoblasts often lie subperiosteally, so the bone piece is transported to the lab in sterile condition using phosphate-buffered solution [Figure 2]a and [Figure 2]b. For osteoblast cells to properly grow in the medium, the following constituents were added: fresh Dulbecco's Modified Eagle Medium, fetal bovine serum (FBS), 50 μg/ml of glutamine solution, 50 μg/ml of gentamycin, and 2 mg/ml of amphotericin B. The transfer medium and growth medium were prepared in the same way except that the transfer medium is serum free.
Figure 2: (a and b) Removal of segment of iliac crest for creating gap nonunion and collection of osteoblasts

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The bone pieces were placed in alpha minimal essential medium (MEM) solution with 10% penicillin and streptomycin. Soft tissue and periosteum were removed through scrapping and extensive washing. The bone samples were washed, and then finally cut into 1–2 mm length. The bone samples were incubated in collagenase solution for 25 min, and then the solution was aspirated and kept for cell plating. This was repeated for two more times, for a total of three digestions.

Osteoblast culture

Cell suspension resulting from primary isolation procedure was cultured in Type I rat tail collagen-coated 6-well plates at a seeding density of approximately 250,000 cells/9.5 cm2 in an alpha-MEM medium supplemented with the following constituents: 5% FBS, 5% calf serum, and 1% penicillin and streptomycin. Cells were maintained at 37°C in a humidified incubator for 7 days.

Cell seeding onto nano-hydroxyapatite/tricalcium phosphate/gelatin/chitosan scaffold

Osteoblasts were grown in batch mode for 30 days to check their viability using an hemocytometer. The cells showed a near-linear growth for 21 days and after the completion of 21 days, there was a decrease in the number of cells.

Bioreactor enhancement

Cell polymer construct is transferred into 110 cm3 volume airlift bioreactor. The vessel is cleaned with 70% ethanol and double-distilled water and autoclaved sterilized (30 min at 121°C). The construct was placed in the airlift bioreactor. The airlift reactor is provided with gas supply. Sterile incubator gas will pump through the riser at a flow rate of 0.7–1.2 L/min. The medium will replace 50% v/v corbon dioxide once per week through continuous gas exchange. The construct will be cultured for 2 weeks in 37°C/10% CO2 incubator [Figure 3].
Figure 3: Cell seeded scaffold

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The cultured autologous osteoblasts over the three-dimensional scaffold were transferred into the defect by reopening the iliac crest of the same rabbit from which cells were extracted. Biocompatibility of the scaffold material was tested in one of the control limbs. The same procedure was repeated in the left limbs without osteoblast transplantation. Thus, the defect in the right iliac crest of each rabbit was transplanted with scaffold impregnated with autologous osteoblasts, and the left iliac crest served as control. The scaffold is cut in such a size that it tightly fits into the defect so that no additional stability/implant is required [Figure 4]. The iliac crest is then closed in layers using aseptic precautions. The rabbits were followed up after a period of 4, 8, 12, and 20 weeks by gross inspection and X-ray examination and histology.
Figure 4: Implantation of cell seeded scaffold on created iliac crest defect in rabbits

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  Results Top

[Table 1] shows gross inspectory findings in the form of uptake of scaffold. A relative scoring has been awarded to the rabbits' skiagram, and the mean was calculated as per the given score in [Table 2].
Table 1: Gross inspectory findings in the form of uptake of scaffold

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Table 2: A relative scoring has been awarded to the rabbits' skiagram and the mean was calculated as per the given score

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  • 0 = No evidence of new bone formation
  • 1 = Little amount of callus formation
  • 2 = All around the margin of the scaffold, there is increase in the radio opacity: Calcification
  • 3 = Formation of bridging mass in the created defect; increased radiodensity
  • 4 = Increase in the girth/density reaching up to the periphery of the created defect.

Based on the observations of relative scoring at different period of follow-up, the Student's t-test (two-tailed test) revealed P = 0.012, which was highly significant.

[Figure 5] and [Figure 6] show X-ray follow-up of two rabbits showing the stages of incorporation of graft. [Figure 7] shows the histology of control site at 20 weeks showing fibrous tissue and poor osteogenesis. [Figure 8] shows good osteogenesis, however scaffold has not fully degraded.
Figure 5: Rabbits followed up showing union of the scaffold in bone gap at the right iliac crest, at final follow-up

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Figure 6: Poor uptake of scaffold and poor bonding

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Figure 7: Photomicrograph showing the histology of control site at 20 weeks showing fibrous tissue and poor osteogenesis

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Figure 8: Photomicrograph at 20 weeks on test limb showing good osteogenesis, however scaffold has not fully degraded

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  Discussion Top

Despite increase in the number of procedures that require bone grafting, there has not been a single ideal bone graft substitute. In light of these inadequacies, numerous investigators, in their search for suitable alternatives to autologous bone, have explored such diverse substances, such as homologous bone (allografts), heterologous bone (xenografts), demineralized bone, deproteinated bone, and synthetically derived organic and inorganic constituents of bone.[9] Yet these alternatives have been largely ineffective in fulfilling the basic mechanisms underlying bone regeneration; vis-à-vis osteogenesis, osteoinduction, and osteoconduction. Of the recent approaches to the tissue engineering approach for healing of bone defect/gap nonunion, human mesenchymal stem cells or adipose derived stem cells (ASC) on scaffolds have been used by various researchers in order to decipher appropriate pathway for osteogenesis by osteoblasts.[10],[11]

Results of the present study demonstrate that new bone formation can be elicited in critical-sized defects in iliac crest of rabbit by the implantation of autologous cultured osteoblast impregnated in a three-dimensional, nanomaterial biodegradable scaffold. Under the conditions used in the present study, the implantation of autologous osteoblasts in HAP scaffold led to the formation of new bone. The new bone was not distributed uniformly throughout the cell–matrix implant but became integrated with the host bone.

Use of nanohydroxyappatite as nanomaterial appears to improve osteogenesis. Results by Lu et al. on nanomaterial and understanding the cell signaling pathway for osteogenesis by ASC), demonstrated that bioactive glass nanoparticle (nBG)-incorporated polycaprolactone coating on HAP/β-TCP (HA/TCP) scaffold exerted a synergistic effect with 3 days of bone morphogenetic protein (BMP-2) treatment in promoting osteogenic gene expression levels (Runx-2, collagen I, osteopontin, and bone sialoprotein) and alkaline phosphatase activity in ASCs. They revealed that the synergistic effect was mediated through a mechanism of activating β1-integrin and induction of Wnt-3a autocrine signaling pathways by nBG-incorporated scaffold.[10] Our study uses a more simplified scaffold by using nano HAP/TCP instead of bioactive glass. In addition, costly BMP-2 treatment was not instilled, but chitosan and gelatin were used for adherence of osteoblast.

Chitosan, being a natural biopolymer, upregulates genes associated with calcium binding and mineralization such as collagen type 1 alpha 1, integrin-binding sialoprotein, osteopontin, osteonectin, and osteocalcin, significantly as demonstrated byin vitro studies by Mathews et al.[12] Thus, osteoblast appears to be directly activated with a pathway mentioned above or a similar one.

Radiology of the implanted iliac crest revealed that there was progressive resorption of the implant, but the implant persisted even at 20 weeks; this finding was also substantiated by gross examination of the specimens and histopathology [Figure 7] and [Figure 8]. Previously, similar studies done using bioactive glass ceramics, carbon fiber glass implants, fiber-composites, and various other ceramic implants did not show any change in the consistency of the implant even after 20 weeks of follow-up, proving that none of the implants were biodegradable.[13],[14]

The biodegradability of the implant was a desired and favorable sign, but the resorption of the implant was not associated with a corresponding new bone formation which could be appreciated radiologically. This discrepancy could be assumed to be due to the failure of the gelatin in the implant to provide the required scaffold for a longer duration.

Human trial in Chinese population using bio-derived bone scaffold with allogenous cultured periosteum-derived human osteoblasts showed achievable union in 3–4.5 months after creation of bone gap and followed till 7 years for any evidence of loosening. However, in the study by Penget al., 60% of fractures of long bones were fresh and comminuted. They could not generalize the results for gap nonunion.[15] Another Chinese trial on 52 patients showed excellent results and union in bone defects followed for a period of 18.5 months by allogenous periosteum-derived osteoblast over a bio-derived bone scaffold, However, the composition of the scaffold was not specified.[16]

The design and selection of an ideal carrier for the delivery of osteoblast cells is based on several criteria. First, the material should allow for uniform loading and retention of cells. Second, the carrier should support rapid vascular ingrowth. Third, the matrix should be composed of radiolucent materials that are resorbed and replaced by bone as new bone is formed. Fourth, the material should allow or enhance osteoconductive bridging of host bone by the new bone. Finally, the cell–matrix combination should be easy for the physician to handle in a clinical setting. The NHAP/TCP/gelatin/chitosan used in this study degrades slowly in the initial hours, and the rate of degradation increases with time. The rate of growth of osteoblast increases with time in culture medium till 21 days when the amount of cell is highest, and this is the time for seeding the cells on scaffold. The viability of the cells in scaffold decreases with time and therefore, it is important to implant the scaffold as early as possible. This will impart a three-dimensional architectural similarity to that of natural bone, thus the scaffold that we implant at non union site will help in the new bone formation. The aligned structure should help the mineralization of the newly formed bone in an oriented fashion, thus hastening the process of bone remodeling at a faster rate. This is due to two broad reasons; first, the time taken for reorientation of the newly formed bone to that of the natural bone texture will almost vanish and second, the presence of NHAP/chitosan at the site of new bone formation will help the healing process of bone regeneration.

  Conclusion Top

Our pilot study on rabbits used autologous osteoblast incorporated on nanomaterial scaffold which did provide a suitable scaffold for homing of bone-forming cells, as evidenced by the callus at the implant–bone interface in a gap nonunion. The nanoparticle properties and chitosan did provide synergistic action for such incorporation.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

Tomlinson J. Complications of fractures repaired with casts and splints. The Veterinary clinics North America: Small animal practice. 1991;21:735-44.  Back to cited text no. 1
Sumner-Smith G, Cawley AJ. Nonunion of fractures in the dog. J Small Anim Pract 1970;11:311-25.  Back to cited text no. 2
Schmitz JP, Hollinger JO. The critical size defect as an experimental model for craniomandibulofacial nonunions. Clin Orthop Relat Res 1986;(205):299-308.  Back to cited text no. 3
Hollinger JO, Kleinschmidt JC. The critical size defect as an experimental model to test bone repair materials. J Craniofac Surg 1990;1:60-8.  Back to cited text no. 4
TRUST Investigators writing group, Busse JW, Bhandari M, Einhorn TA, Schemitsch E, Heckman JD, et al. Re-evaluation of low intensity pulsed ultrasound in treatment of tibial fractures (TRUST): Randomized clinical trial. BMJ 2016;355:i5351.  Back to cited text no. 5
Thackray AJ, Taylor J. Towards evidence-based emergency medicine: Best BETs from the Manchester Royal Infirmary. BET 2: Immobilisation of stable ankle fractures: Plaster cast or functional brace? Emerg Med J 2013;30:513-4.  Back to cited text no. 6
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Quarto R, Mastrogiacomo M, Cancedda R, Kutepov SM, Mukhachev V, Lavroukov A, et al. Repair of large bone defects with the use of autologous bone marrow stromal cells. N Engl J Med 2001;344:385-6.  Back to cited text no. 8
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Zeitouni S, Krause U, Clough BH, Halderman H, Falster A, Blalock DT, et al. Human mesenchymal stem cell-derived matrices for enhanced osteoregeneration. Sci Transl Med 2012;4:132ra55.  Back to cited text no. 11
Mathews S, Gupta PK, Bhonde R, Totey S. Chitosan enhances mineralization during osteoblast differentiation of human bone marrow-derived mesenchymal stem cells, by upregulating the associated genes. Cell Prolif 2011;44:537-49.  Back to cited text no. 12
Cortez PP, Silva MA, Santos M, Armada-da-Silva P, Afonso A, Lopes MA, et al. A glass-reinforced hydroxyapatite and surgical-grade calcium sulfate for bone regeneration:In vivo biological behavior in a sheep model. J Biomater Appl 2012;27:201-17.  Back to cited text no. 13
Knowles JC, Hastings GW, Ohta H, Niwa S, Boeree N. Development of a degradable composite for orthopaedic use:In vivo biomechanical and histological evaluation of two bioactive degradable composites based on the polyhydroxybutyrate polymer. Biomaterials 1992;13:491-6.  Back to cited text no. 14
Peng K, Xiang Z, Yang Z, Xie H, Wu X, Huang F, et al. Clinical application of bio-derived bone transplantation with tissue engineering technique: 7 year follow-up. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi 2008;22:606-9.  Back to cited text no. 15
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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]

  [Table 1], [Table 2]


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