Cold physical plasma treatment optimization for improved bone allograft processing

Abstract

In musculoskeletal surgery, the treatment of large bone defects is challenging and can require the use of bone graft substitutes to restore mechanical stability and promote host-mediated regeneration. The use of bone allografts is well-established in many bone regenerative procedures, but is associated with low rates of ingrowth due to pre-therapeutic graft processing. Cold physical plasma (CPP), a partially ionized gas that simultaneously generates reactive oxygen (O₂) and nitrogen (N₂) species, is suggested to be advantageous in biomedical implant processing. CPP is a promising tool in allograft processing for improving surface characteristics of bone allografts towards enhanced cellularization and osteoconduction. However, a preclinical assessment regarding the feasibility of pre-therapeutic processing of allogeneic bone grafts with CPP has not yet been performed. Thus, this pilot study aimed to analyze the bone morphology of CPP processed allografts using synchrotron radiation-based microcomputed tomography (SR-µCT) and to analyze the effects of CPP processing on human bone cell viability and function. The analyzes, including co-registration of pre- and post-treatment SR-µCT scans, revealed that the main bone morphological properties (total volume, mineralized volume, surface area, and porosity) remained unaffected by CPP treatment if compared to allografts not treated with CPP. Varying effects on cellular metabolic activity and alkaline phosphatase activity were found in response to different gas mixtures and treatment durations employed for CPP application. It was found that 3 min CPP treatment using a He + 0.1% N₂ gas mixture led to the most favourable outcome regarding a significant increase in bone cell viability and alkaline phosphatase activity. This study highlights the promising potential of pre-therapeuthic bone allograft processing by CPP prior to intraoperative application and emphasizes the need for gas source and treatment time optimization for specific applications.

Keywords: allografts; cancellous bone; cold atmospheric pressure plasma; mesenchymal stromal cells; plasma medicine; synchrotron radiation computed tomography.

FIGURE 1

Preparation of allogeneic bone scaffolds. (A) Shown is a plate of trabecular allogeneic bone with a height of 5 mm after rehydration. (B) Cylindric pieces with a diameter of 6 mm were punched out from the plate. (C) The CPP flame is able to penetrate porous allogenic bone scaffolds. (D) No bacterial growth was found after 24 h, thus confirming sterility of the CPP treated scaffolds. (E) CPP treatment (t2) was performed in a multi-well plate using a clinical-grade device (kINPen). (D: Student’s t-test, paired, n = 3).

FIGURE 2

SR-µCT prior to CPP treatment for basic scaffold characterization. (A) Shown are the cropped regions of the reconstructed volumetric µCT data prior to CPP treatment (t1). (B) Tb.Th and Tb.Sp were determined prior to CPP treatment (t1) for all scaffolds. (Student’s t-test, unpaired, n = 3, *p < 0.05).

FIGURE 3

The influence of CPP treatment on morphological parameters of allogeneic bone. (A) Shown are morphological bone parameters for control and CPP treatment group. SR-µCT scans were performed prior to (t1, white bars) and after (t2, black bars) CPP treatment. (B) Shown are the values of the respective morphological parameters after normalization of t1 to t2. (C) Registration of volumetric reconstructions at t1 and t2 allows for the detection of TV loss (blue) and gain (green). The differences between t1 and t2 did not exceed the expected registration error. [(A,B) Student’s t-test, paired, n = 3].

FIGURE 4

Optimisation of CPP gas source and treatment time prior to seeding with human mesenchymal stromal cells. (A) Scaffolds were treated by CPP using different carrier gases. The CPP treated scaffolds where then seeded with mesenchymal stromal cells, followed by investigation of metabolic activity after 4 and 7 days (B) Carrier gas sources were refined and CPP treatment time was prolonged to 3 min. After subsequent cell seeding, metabolic activity was determined after 4 and 7 days. (C) Based on previous results, CPP treatment time was prolonged to 5 min while only 5 carrier gases were tested. Metabolic activity of mesenchymal stromal cells seeded onto the CPP-treated scaffolds was determined after 4 and 7 days. [(A–C) one-way ANOVA, multiple comparisons, n = 6, *p < 0.05, **p < 0.005].

FIGURE 5

Optimisation of CPP gas source and treatment time to improve osteogenic function of human mesenchymal stromal cells (hMSCs). (A) Shown is the ALP activity as a measure of osteogenic function after CPP treatment for 1 min with varying gas sources. (B) Since viability was found to be reduced when Ar based gas sources are used, a 3 min treatment time was performed using an He based carrier gas. (C) After prolonging the treatment time to 5 min, hMSCs were seeded onto the scaffolds and their ALP activity was determined after 7 days. [(A–C) one-way ANOVA, multiple comparisons, n = 6, *p < 0.05].