Percutaneous autologous stem cell enriched marrow concentrate injection for treatment of cases of impaired fracture healing with implant in situ: A cost-effective approach in present Indian scenario

Inclusion and exclusion criteria

The inclusion criteria were as follows: (i) Patients of either sex, 18-65 years of age, (ii) radiographic evidence of disturbed fracture union (with no progression of fracture healing over 3 consecutive months) with the implant in situ. Non progression of fracture healing was further defined on radiographs by: (i) absence of bone crossing the fracture site, (ii) persistent fracture line(s), and (iii) lack of evidence of progression of healing on serial imaging.

The exclusion criteria were as follows: (i) Active infection, (ii) active inflammatory or connective tissue disease (i.e., lupus, RA); (iii) any medical comorbidity such as cardiac or pulmonary disease, endocrine disorder; (iv) hypersensitivity to nonsteroidal anti-inflammatory drugs (NSAIDs), abnormal liver or kidney function tests, history of peptic ulceration and upper gastrointestinal hemorrhage, cancer, and hyperkalemia; (v) history of coagulopathies and hematological or neurological disorders; (vi) and/or any other conditions that in the our opinion, would make participation not in the best interest of the patient or could prevent or limit the protocol-specified outcome assessment.

Study design

The study was designed as a single-center prospective research. The steps were as shown in Figure 1.

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Figure 1

Study design: Schematic flow chart showing different steps

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Device description

The present study utilized a REMI centrifuge C-854/6 System [Figure 2].

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Figure 2

REMI centrifuge C-854/6 System

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Methodology

Before conducting the procedure, osseous defect was measured approximately by X-rays in two planes (anteroposterior (AP) and lateral view).

Aspiration of bone marrow

With all aseptic precautions under local anesthesia (1% lidocaine without epinephrine) in prone position, a small 2-mm skin incision was made with a sharp pointed scalpel blade over posterior iliac crest. The aspiration needle (16G) is pointed anterolaterally in the direction of the iliac crest and was advanced slowly through the incision. The beveled needle was securely placed within the marrow cavity/spongy bone;^[15] the marrow aspirated into a Dispovan 20 ml plastic syringe (heparin rinsed). At a given depth, the needle is turned 45° during successive aspirations to reorientate the bevel, thereby facilitating aspiration from larger space. After the tip is swept around a full circle, the needle was withdrawn approximately 1 cm proximally and successive aspirations were begun, repeating the same procedure.

Processing and density gradient centrifugation

We employed Ficoll-Paque technique of density gradient separation for the isolation of mononucleated cells. As this technique takes advantage of the density differences between mononuclear cells and other elements found in the blood sample, it yielded a virtually Red blood cells (RBCs)-free bone marrow cell suspension as described by Wells et al.^[16] Heparinized marrow was diluted 1:1 in phosphate-buffered saline (PBS) and carefully layered over the Ficoll-Paque gradient in approximately 1:2-2.5 ratio (Ficoll-Paque solution of 1.077 g/ml density; ~1:2.5^[17]). The bone marrow aspirate was then concentrated using the centrifugation system (REMI centrifuge C-854/6) [Figure 2] for 10 min at 500 g (~2,000 rpm) in the operating room at room temperature. The majority of the mononucleated cells and platelets accumulated on top of the Ficoll-Hypaque layer because they have a lower density; in contrast, RBCs and granulocytes have a higher density than Ficoll-Hypaque and sediment at the bottom of the Ficoll-Hypaque layer [Figure 3]. The mononuclear cell interface layer was harvested carefully using long bore micropipette after discarding the supernatant. The resultant mononuclear cells were resuspended twice in PBS (for 5 min at 500 g (~2,000 rpm) [Figure 4]. This technique reduces 120 ml of bone marrow aspirate to a 'concentrated myeloid' suspension of about 6-7 ml. The concentrate transferred by sterile technique back to the sterile field for reinjection. Both aspirate and concentrate (1 mL were saved of each sample for in vitro analyses) were analyzed using flow cytometry (fluorescence activated cell sorting (FACS)) regarding the containing cells [Figure 4]. One drop of concentrate is taken on the slide for staining with trypan blue dye (1:1) to assess the viability of mononuclear fraction. Sample is also sent for culture and sensitivity test. Rest is preserved for injection under IITV control. The concentration factor was calculated cell count-quotient bone marrow aspirate concentrate (BMAC)/aspirate after the centrifugation procedure.

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Figure 3

Showing Density gradient sedimentation. (a) RBCs and granulocytes; (b) clear layer of Ficoll; (c) buffy colored mononuclear fraction; and (d) clear layer of plasma. RBC = Red blod cell

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Figure 4

(a) Buffy colored concentrate of mononuclear fraction after washing (twice) with PBS and (b) a magnified view. PBS = Phosphate-buffered saline

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Injection of BMAC (in vivo application)

The diluted BMAC (2 ml) is injected slowly at a defect site. After the injection, the needle is gradually withdrawn, with small oscillating motions being made to fill in the path of the needle.

Postprocedure protocols

Drugs: Analgesic (for 3 days and then Si opus sit), antacid (for 3 days), antibiotics (for 3 days), and supplements (calcium and multivitamins). Immobilization: 4-6 weeks. Physiotherapy: Active, assisted physiotherapy and mobilization of adjacent joints. Weight bearing: Partial weight bearing (toe-touch): Week 6-10 and full weight bearing: Week 10-12 (considering compliance of the patient and bone quality; clinical and radiological parameters). X-ray assessment (at least two planes): To record the orientation of implants, and also it provides a basis for the evaluation of how fracture healing is progressing.

Criteria for evaluation of outcome

Statistical analysis

The data are presented as means ±standard deviation (SD). All calculations and statistics were performed with Statistical Package for Social Sciences (SPSS 20) software. A P - value of less than 0.05 (P < 0.05) was regarded as significant. The internal consistency (reliability) of the scale was assessed using Cronbach's alpha coefficient.

From Indian perspectives

In developing countries like India, the most important factor in fracture healing is affordability.^[54] In developing world, impaired fracture healing has profound human and socioeconomic consequences in all societies. The economic effects of fracture healing impairment can be divided into direct and indirect costs.^[55,56] Furthermore, the psychological and physical trauma to the patient when faced with the prospect of another surgery is often underestimated. Although no hospital-specific data are available, any multispecialty hospital treats almost 500-700 cases of impaired fracture healing per year.^[57] There are number of such tertiary care centers in each metropolitan city, hence India has to deal with an enormous burden of complicated fractures.^[54,57] If we aim at providing maximum relief to the largest number we will have to develop and adopt techniques to suit our requirements.^[58] The developed countries do not have to conduct research for our clinical problems. Mere importation of sophisticated, expensive equipments and methodology; however, does not solve the problem. We have to find out solutions to our problems as well as devise cost-effective problem-solving strategies to one and all.^[59,60]

For these reasons, an impetus exists to develop and validate alternative cost-effective technique in developing country contexts using simple laboratory centrifugation system and locally available equipments/reagents that is capable of replicating the performance of the published literatures significantly, while dramatically reducing the cost (both tangible and intangible), complexity of sophisticated procedure, and is accessible and affordable to mass population.

In our setting, the proposed technique enriched population of bone marrow-derived cells by a factor of 7.82 (before 16.55 ± 0.5127/μL; after centrifugation viable CD34 = 129.55 ± 10.01513/μL (P < 0.05)) [Figure 4]. Consequently, concentration capacity of the current centrifugation system was more efficient than the enrichment factor of published literature [Table 2]. Furthermore, we strongly support Kasten et al., who reported higher MSC yields [Table 2] owing to effective isolation technique (SEPAX VOL RED protocol) of the MSCs.^[12] In agreement with previous studies,^[8,61] in present pilot study cellular concentration showed considerable individual variations (range 1.98-19.1); probably due to physiological variables (age, menopause), associated co-morbidities (smoking), or technical reasons/variables. The authors also agreed with the fact that the concentration of osteoprogenitor cells may vary significantly between individuals, aspiration sites, aspiration technique, and isolation protocols.^[8,12,61] In the current study, flow cytometric analysis [Figure 8] were done just to determine the subset of circulating cells expressing the CD34 antigen to assess the progenitor cell content in the whole marrow and after density gradient centrifugation, that is, in concentrate (the facility for CD specific for MSCs are not available; in view of that we used CD34 just to assess the efficiency of concentration). Alternation in preservation or processing of bone marrow concentrate significantly reduces the number of viable CD34 + cells available for reinfusion^[62] as also noted in the present series (viability of fresh cells was 94.7 ± 3.6%; in vitro analysis showed a viability level of 75.7 ± 9.16%; P < 0.05). An immediate autologous injection of bone marrow concentrate dramatically improves the quality of cells as it significantly reduces the complications pertaining to preaging, decreased cell viability, or dedifferentiation that are usually associated with in vitro cultivation.^[63,64] Therefore, "one-step" isolation/application of MSCs enriched fraction with on-table preparation was advocated, as it increases the viability of cells while reducing the cost and infection rates by decreasing the ex vivo time period.

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Figure 8

Flow cytometric analysis of aspirate (a) and concentrate (b); showing significant concentration of mononuclear fraction (CD34, CD45, and lymphs)

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The result from existing prospective study strongly supported previous studies'^{[4,9,10,11,64]} [Table 2] concepts that centrifugation increases the efficiency of bone marrow owing to increase the concentration of osteoprogenitor cells.

Defect or distance between the fracture ends is a critical factor for success of healing; a distance of 5 mm or less between the fractures ends resulted in healing in all cases except one in present series. Previous study^[4] has also reported a significant association between fracture gap and outcome. The one unsuccessful case had bone defect greater than 5 mm and open bone grafting was necessary within the follow-up period.

In current study, in an average of 12.28 (SD1.38) weeks, a bony union was achieved in seven of eight patients [Table 1]. This finding is consistent with that of Hernigou et al.^[4] It has also been noted that patients who received bone marrow concentrate injection experienced a lower time to union [Table 9]. This efficacy can be attributed to the implantation of increased concentration of osteoprogenitor cells. The number and concentration of the stem cells being injected, affected the clinical outcome.^[4] Greater the concentration of osteoprogenitor cells, less will be the healing period.

Table 9 Effectiveness of bone marrow aspirate/bone marrow aspirate concentrate injections for impaired fracture healing

Based on the critical data analysis of present series, it was found that there was no significant relationship (P > 0.05) among demographic variables (e.g., age and sex) [Table 4] and treatment outcome; as also concluded by Hernigou et al.^[4] This indifference indicates a common etiologic basis, that is, locally decreased population of progenitors.^[68] Although the location of the fracture appeared in earlier study^[6] to influence the healing, we were unable to confirm this [Table 4]. One fundamental reason for this issue is smaller sample size and other could be attributed almost entirely to the existing biomechanical environment.^[68,69] Furthermore, patients with comorbidities (smoking) took a longer time to heal (average 14 weeks) than other patients (11.6 weeks (SD 0.89); P < 0.05) [Table 3]. Several studies^[70,71,72] have shown that consolidation of a fracture is often delayed in heavy smokers and drinkers owing to overall reduction in the numbers of progenitor cells in the bone marrow. In the present series, there were only two smokers [Table 1]. Consequently, the low rate of smokers led to the low mean value in present series, probably resulting in a lack of correlation between outcome and smoking behavior.

Most of published techniques^{[4,9,10,11,12,17,64]} need special, sophisticated devices; but in practice, high logistical requirements and exorbitant cost of these commercially available kits restricts their widespread use. Availability has been limited to research centers, and it remains quite expensive. For this reason the present research is currently focused on the development of methodology that is feasible with low-cost devices. The critical analysis reveals a large difference in costs of hardware device of present study and others [Table 10]. Furthermore, the device used in the present study [Figure 2 and Table 10] is easily available even at second and third tier of Indian three-tier system^[68,73] of health care's.

Table 10 Approximate cost of the device used (does not include any other price)

Patient satisfaction is an integral part of modern quality management. Treating surgeon should analyze the patient's expectations with respect to surgery: Labors, farmers, and office or sedentary workers; all have different needs. Therefore, a doctor's treatment protocol should not only be limited to objective measures but also include background information about the patient's lifestyle and profession (subjective measures).

Although many authors have reported on patient satisfaction after arthroplasty,^{[74,75,76,77]} we are unaware of any validated self-report scale that evaluates patient satisfaction after percutaneous BMAC injection. In our setting, the majority of responses were in the very satisfied or somewhat satisfied categories [Table 5]. During the follow-up period after 6 week, more patients (87.5%) were satisfied with pain relief, their mental health and social functioning, and their ability to do daily activities compared to improvement in their ability to do professional activities [Table 5]. Further, it was found that the total satisfaction scores were gradually increased over the subsequent follow-up visits (85.7%: very satisfied and 14.28%: Somewhat satisfied) [Table 5].

In the present study, one out of eight patients [Table 5] is somewhat dissatisfied following proposed intervention. Potential reasons for patient dissatisfaction with intervention may be poor mental health or unfulfilled expectations (a further research study will also help to find out the causes for dissatisfaction). In present series, Cronbach's alpha coefficient for internal consistency of the satisfaction scale is 0.93 at 6 week and 1.0 at 1 year of follow-up, suggesting excellent reliability [Table 6]. In conclusion, present satisfaction scale may be used to evaluate the complex relationship between patient's baseline parameters (items) with outcome.

In present series, there has been no hematoma formation, or infection at the site of aspiration. None of the patients in the current study reported chronic pain at aspiration site. In none of the cases we had to repeat the procedure to achieve union. Aspiration of bone marrow from the iliac crest has not been a "rate-limiting" factor in rehabilitation or a risk factor for prolonged hospital stay.

In context with the present cohort and diamond model for bone-fracture healing,^[77] it is an arduous task for to speculate as to whether or not BMAC is an effective therapy in cases of unstable fracture with impaired healing process. Furthermore, any scaffold has not been used in present study. In fact, it is anticipated that the use of needle during the procedure to deliver the BMAC to the target site seems to be traumatic enough to create a whole marrow scaffold.

Limitations

Whilst the findings of the current study could be applied in most instances, there were some important limitations.

1. It is unclear whether only some or all stem cells in the concentrate differentiated into osteoblasts and integrating normally once injected. It is also difficult to tell that how many stem cells are required to induce bone regeneration in vivo.

2. The present study is nonrandomized and does not include any control groups and small sample size further limits the generalization of findings of present study.

3. Likert response scale yields ordinal rather than interval data.

Source of Support:

Nil