Platelet-rich plasma enhances rib fracture strength and callus formation in vivo

Abstract

BACKGROUND:

Rib fractures are a common traumatic injury affecting more than 350,000 patients a year. Early stabilization has shown to be effective in reducing pulmonary complications. Platelet-rich plasma (PRP) is a growth factor–rich blood product known to improve soft tissue and bone healing. We hypothesized that the addition of PRP to a rib fracture site would accelerate callus formation and improve callus strength.

METHODS:

Platelet-rich plasma was isolated from pooled Lewis rat blood and quantified. Thirty-two Lewis rats underwent fracture of the sixth rib and were treated with 100 μL PRP (1 × 10⁶ platelets/μL) or saline. At 2 weeks, ribs were harvested and underwent a 3-point bend, x-ray, and microcomputed tomography, and callus sections were stained with 4′,6-diamidino-2-phenylindole and Alcian blue and picrosirius red. At 6 weeks, ribs were harvested and underwent a 3-point bend test, x-ray, microcomputed tomography, and Alcian blue and picrosirius red staining.

RESULTS:

At 2 weeks, PRP increased callus diameter (9.3 mm vs. 4.3 mm, p = 0.0002), callus index (4.5 vs. 2.1, p = 0.0002), bone volume/total volume (0.0551 vs. 0.0361, p = 0.0024), cellularization (2,364 vs. 1,196, p < 0.0001), and cartilage (12.12% vs. 3.11%, p = 0.0001) and collagen (6.64% vs. 4.85%, p = 0.0087) content compared with controls. At 6 weeks, PRP increased fracture callus diameter (5.0 mm vs. 4.0 mm, 0.0466), callus index (2.5 vs. 2.0, p = 0.0466), BV/TV (0.0415 vs. 0.0308, p = 0.0358), and higher cartilage (8.21% vs. 3.26%, p < 0.0001) and collagen (37.61% vs. 28.00%, p = 0.0022) content compared with controls. At 6 weeks, PRP samples trended toward improved mechanical characteristics; however, these results did not reach significance (p > 0.05).

CONCLUSION:

Rib fractures are a common injury, and accelerated stabilization could improve clinical outcomes. Platelet-rich plasma significantly increased callus size, calcium deposition, and cartilage and collagen content at 2 and 6 weeks and trended toward improved strength and toughness on mechanical analysis at 6 weeks compared with controls, although this did not reach significance. These findings suggest that PRP may be a useful adjunct to accelerate and improve fracture healing in high-risk patients.

Rib fractures are the most common skeletal thoracic injury and occur in approximately 10% of trauma patients.^1,2 Rib fractures cause pain-limited hypoventilation, disrupt breathing efficiency, and impair gas exchange due to associated lung damage.³ These factors lead to increased incidence of pneumonia, respiratory failure, and increases in mortality. Early surgical stabilization of rib fractures has improved outcomes in patients with flail and nonflail rib fractures yet carries an inherent operative risk and may not be indicated in all patients.⁴ Thus, many patients suffer through nonoperative management predicated on pulmonary toilet and judicious pain control. Because of advancements in regenerative medicine, our understanding of the underlying mechanisms of rib healing, and thoracic surgical technology, this paradigm is being actively re-examined and adjuncts to accelerate fracture healing are sought.

Platelet-rich plasma (PRP) is a growth factor–rich blood product made up of super-concentrated platelets.⁵ Platelet releasates have been shown to increase cellularization and neovascularization of surgical implants, leading to long-term improvements in wound strength and integrity in a dose-dependent fashion.^6–10 Furthermore, super-concentrated platelets have demonstrated enhanced implant fusion and osteogenesis in a rabbit posterolateral spinal fusion model, with earlier bone deposition and maturation.¹¹ Super-concentrated platelets have been shown to modulate the immune system and preferentially promote the increased expression of proteins associated with a regenerative rather than inflammatory phenotype, factors that lead to greater mechanical strength of the wound scar.^12,13 In addition, PRP has been shown to improve the body’s capacity for soft-tissue and cartilaginous healing via acceleration of cell proliferation and angiogenesis in the settings of osteoarthritis and rotator cuff injuries.^14–17 In animal models of osteoarthritis, super-concentrated platelets demonstrate accelerated recruitment of chondrocytes and synoviocytes to the areas of damage with concurrent upregulation of anti-inflammatory cytokines and increased collagen deposition.¹⁵

Patients with risk factors for poor healing such as hemorrhagic shock, tobacco use, and alcohol use disorder may benefit from adjuncts, such as PRP, to support proper rib fracture healing. While both single growth factor delivery and super-concentrated platelets have been shown to improve bone healing in femurs, what is unknown is whether growth factor–rich concentrated platelets in PRP can achieve early and enhanced fracture healing in ribs.¹⁸ Here, we sought to study the effects of PRP on rib fracture healing in a rat rib fracture model. We hypothesized that the addition of PRP to the fracture site would accelerate callus formation and improve ductility or resistance to fracture.

MATERIALS AND METHODS

Animal Model

Thirty-two adult Lewis rats were purchased from Charles River (Wilmington, MA). Lewis rats were chosen for their inbred nature to allow for a controlled and validated study population and homogenous source of PRP, and they were housed in our institution’s vivarium. Rats underwent rib fracture surgery with the immediate application of PRP or saline for control. Equal number of saline and controls was performed each day to reduce confounders. The surgeon was aware of which animals received which treatments, but analysis was performed in a blinded fashion. Sixteen rats were sacrificed at 2 weeks (8 PRP and 8 saline control) postsurgery, and hemithoraces were removed and then evaluated with x-ray. Half of the rats in each group underwent mechanical testing of their explanted ribs, and the other half underwent microcomputed tomography (micro-CT) followed by staining. This was repeated at 6 weeks with the same number of rats per group. The study was approved by the Institutional Animal Care and Use Committee at our institution (A122–20-06), and all investigators complied with the National Research Council’s Guide for the Care and Use of Laboratory Animals. The experiments in this study were performed in accordance with the animal research: reporting of in vivo experiments 2.0 guidelines (Supplemental Digital Content, Supplementary Data 1, http://links.lww.com/TA/D980).

Platelet-Rich Plasma

Lewis rats were used for PRP harvesting by direct cardiac puncture under deep anesthesia. Briefly, anesthesia was induced within a plastic enclosure via inhaled isoflurane/oxygen mixture at a concentration of 4.5% to 5% and maintained at 2.5% to 4% via nosecone. While supine with the anterior chest fur clipped, the chest wall was sterilized and draped in standard fashion, and the chest cavity was entered sharply at the level of the xyphoid. A 18-gauge needle was used to puncture the right ventricle, and blood was slowly aspirated into 12-mL syringes prefilled with 1 to 2 mL of acid citrate dextrose anticoagulant. Average blood volume yield from a single rat was 8 to 12 mL.

Platelet-rich plasma was isolated from this whole-blood sample via a double-centrifugation technique. Platelet-rich plasma from multiple rats was pooled to allow for adequate platelet quantity. To isolate plasma fractions, the whole blood was first spun at 200g for 15 minutes. Following RBC and buffy coat component removal, the remaining plasma was spun a second time at 1,600g for 10 minutes to isolate the platelets. The resulting pellet was separated from platelet-poor plasma. Platelet counts were quantified using a Multisizer Coulter Counter (Beckman Coulter, Pasadena, CA) and diluted appropriately with plasma to a standardized final dose concentration of 106 platelets per microliter of plasma. The PRP was then stored at −80°C until it was thawed for experimental use. Prior to surgical application, an effective dose of 100 μL of PRP was used, and platelet activation was achieved with 20 μL of 10% calcium chloride as well as subsequent interactions with both exposed collagen I and endogenous tissue factors in the fracture bed.¹⁰

Rib Fracture Surgery

Preoperative analgesics buprenorphine (0.05 mg/kg) and carprofen (5 mg/kg) were administered prior to anesthesia. After appropriate anesthesia was induced with isoflurane as described previously, rats were placed in left lateral decubitus position, and the right lateral chest was clipped of hair, prepared, and draped in standard fashion. A 1.5- to 2.0-cm skin incision is made longitudinally along the sixth rib, and chest wall muscles are bluntly separated to achieve exposure of the sixth rib without violating the pleura. A 6–0 Prolene horizontal mattress stitch is placed in the external oblique muscle, and the sixth rib is sharply divided with wire clippers and displaced. At this time, activated PRP or saline is applied to the fracture site using a pipette. The horizontal mattress stitch is then tied down, and the skin incision is closed with surgical clips. Local anesthesia is then administered via direct application of 25% bupivacaine to the incision. Postoperative analgesia was achieved with administration of buprenorphine (0.03 mg/kg) and carprofen (5 mg/kg), which was also administered daily until postoperative day three. Throughout postoperative week 1, the rats were weighed and examined daily for any signs of hypoxia, bleeding, infection, hematoma/seroma formation, and incision dehiscence. Animals who lost more than 10% weight were given saline injections and moist pellets. On postoperative day 7, surgical clips were removed under anesthesia.

Radiologic Evaluation

Explanted hemithoraces were radiographed with the MultiFocus X-ray Imaging System (Faxitron, Tuscon, AZ). Callus diameter was measured from radiographs, and callus index was calculated by dividing callus diameter by unaffected rib diameter. Rib fracture samples were assessed prior to decalcification with a VivaCT 80 scanner (Scanco Medical, Brüttisellen, Switzerland) using a 55-kVp source. Mineralized volume fraction Bone Volume/Total Volume (BV/TV) was used to quantify calcified callus volume. The point of maximal callus diameter was selected as the callus midpoint, and the midpoint allowed for 300 slices to be selected before and after that point for a total of 600 slices. One threshold value in the system included all original bone and newly formed from the callus. Another threshold value was used to include only the original bone. The BV/TV values from these two analyses then calculated the callus BV/TV.

Mechanical Testing

For mechanical testing, a DMA 850 (TA Instruments, New Castle, DE) with a 10-mm 3-point bend attachment was used. Ribs were placed on the stage with the midpoint of the attachment at the site of fracture if that site could be identified. The ribs were placed concave for testing, if possible; if not, they were placed on their side for relevance of application.

The specimen was stressed at 1 N/min until failure or 18 N (the max force the DMA can apply). Representative curves were used for graphical representation, but samples were testing in at least n = 5 sample size. Flexural modulus of the sample was obtained from the slope of the stress-strain curve at the initial strain values (0–3% strain). Flexural strength values were obtained from the point of the inflection on the stress-strain graphs. This was assumed to be where the rib broke, and the values after this point were where the sample was held together by residue tissue. This point was also used to determine the strain at break of the samples.

Histology and Quantification

After radiographs and micro-CTs were obtained, fracture calluses were formaldehyde fixed for 1 to 3 days, decalcified with ethylenediaminetetraacetic acid, processed for paraffin sectioning using an automated tissue processor. Serial sections (5 μM) were collected with a microtome (Leica, Deer Park, IL) for hematoxylin and eosin staining and immunohistochemical analyses. To quantify cellularization, cellular nuclei were stained with 4′,6-diamidino-2-phenylindole (1:1,000) and quantified using ImageJ (National Institutes of Health, Bethesda, MD). Five fields of view were taken, and then each measured five times to generate 25 independent quantifications for each callus. These were then averaged to determine callus cellularization. To quantify callus cartilage content, calluses were stained with Alcian blue (Sigma-Aldrich, St. Louis, MO) and quantified using ImageJ. Image color channels were split, and the red channel was selected. Identical image thresholds were set to highlight the Alcian blue stain within the callus alone excluding both soft tissue and bone. Particles were then analyzed, and the percentage area staining positive was recorded. To quantify collagen deposition, calluses were stained with picrosirius red (Invitrogen, Carlsbad, CA); same procedure was followed for picrosirius red quantification, except using the blue channel.

Statistical Analysis

The number of rats needed was calculated to attain a power of 0.80 and statistical significance of 0.05. To evaluate rib fracture healing, we collected and analyzed quantitative data on cellularization, callus formation, and mechanical strength at fracture sites at set intervals. We applied paired t tests to compare healing parameters within subjects treated with and without PRP, addressing the paired nature of our data. For multiple group comparisons, we used analysis of variance to identify significant differences in healing outcomes across various PRP concentrations, treatment timings, or other variables. For nonparametric, multiple group comparisons, we used the Kruskal-Wallis test. We used GraphPad Prism 9 (GraphPad Software, Boston, MA) for all statistical analysis. A p value of <0.05 was considered statistically significant.

RESULTS

PRP Increases Rib Fracture Callus Size and Calcium Deposition

Compared with controls, PRP application significantly increased rib fracture callus diameter (9.3 mm vs. 4.3 mm, p = 0.0002) and callus index (4.5 vs. 2.1, p = 0.0002) after 2 weeks. Platelet-rich plasma rib fracture calluses also demonstrated increased calcium deposition on micro-CTwith higher callus mineralized volume fraction (0.0551 vs. 0.0361, p = 0.0024). At 6 weeks, PRP application significantly increased rib fracture callus diameter (5.0 mm vs. 4.0 mm, p = 0.0466) and callus index (2.5 vs. 2.0, p = 0.0466) as seen on x-ray (Fig. 1). In addition, PRP rib fracture calluses at 6 weeks demonstrated increased calcium deposition on micro-CTwith increased callus mineralized volume fraction (0.0415 vs. 0.0308, p = 0.0358) (Fig. 2).

Figure 1.

At 6 weeks, PRP-treated rib fractures demonstrated increased callus diameter and callus index on radiographs compared with controls.

Figure 2.

At 6 weeks, PRP-treated rib fracture demonstrated increased mineralized bone volume on micro-CT compared with control ribs.

PRP Increased Rib Fracture Cellularization and Cartilage and Collagen Deposition

At 2 weeks, rib fractures that received PRP demonstrated higher cellularization than those of controls (2,364 vs. 1,196, p < 0.0001). At 2 weeks, Alcian blue staining showed higher cartilage content in the PRP group calluses compared with control calluses (12.12% vs. 3.11%, p = 0.0001). Picrosirius red stains showed higher collagen content in PRP rib fracture calluses (6.64% vs. 4.85%, p = 0.0087) (Fig. 3). At 6 weeks, Alcian blue staining of rib fractures that received PRP demonstrated higher cartilage content compared with control calluses (8.21% vs. 3.26%, p < 0.0001). At 6 weeks, Picrosirius red staining of rib fractures that received PRP demonstrated higher collagen content than control calluses (37.61% vs. 28.00%, p = 0.0022) (Fig. 3).

Figure 3.

At 6 weeks, PRP-treated rib fracture demonstrated increased cartilage and collagen deposition suggestive of increased endochondral ossification.

PRP’s Effect on Mechanical Strength of Rib Fracture Callus

At 2 weeks, there was no statistically significant difference between PRP-treated ribs (5.66E7 [1.42E7–1.42E8] Pa) and control ribs (4.52E7 [1.95E7–7.47E7] Pa) in toughness (Fig. 4, p > 0.9999). There was no significant difference between PRP-treated ribs (4.69E4 [1.37E4–7.45E4] Pa) and control ribs (7.05E4 [8.52E3–1.34E5] Pa) in flexural modulus (Fig. 4, p = 0.977). The PRP-treated samples (1.89E6 [1.21E6–2.99E6] Pa) had a trend toward lower flexural strength compared with control fractures (3.09E6 [1.84E6–7.37E6] Pa) (Fig. 4, p = 0.8059).

Figure 4.

At late time points, PRP-treated ribs trended toward increased toughness and strength, although this did not reach significance.

At 6 weeks, there was no statically significant difference between PRP-treated ribs (8.015E7 [7.37E7–2.53E8] Pa) and control ribs (5.40E7 [4.18E7–8.26E7] Pa) in toughness (Fig. 4, p > 0.9999). There was no significant difference between PRP-treated ribs (1.56E8 [2.40E7–2.71E8] Pa) and control ribs (2.18E7 [1.32E7–7.51E8] Pa) in flexural modulus (Fig. 4, p = 0.9942). There was no significant difference between PRP-treated ribs (2.19E7 [6.45E6–3.05E7] Pa) compared with controls (6.40E6 [4.14E6–2.84E7] Pa) in flexural strength (Fig. 4, p > 0.9999).

DISCUSSION

The effects of PRP application on rib fracture healing in a rat model were investigated using radiographic, histologic, and mechanical evaluation. Radiographically, PRP-treated fractures exhibited significantly larger calluses and had more calcium deposition compared with controls at both early and late time points. Histologically, PRP-treated ribs demonstrated increased cellularity at 2 weeks and increased cartilage and collagen deposition at both early and late points. Regarding mechanical testing, no major difference was observed between control and PRP-treated samples at 2 weeks, despite enhancement of maximum strain after the initial break. At 6 weeks, PRP-treated ribs demonstrated a trend toward higher toughness, flexural modulus, and flexural strength than control samples, although this did not achieve significance. Taken together, these findings support the hypothesis that PRP application at the time of fracture accelerates rib fracture healing and results in stable and mature calluses with increased cartilage deposition and enhanced endochondral ossification.

Our data suggest that, by administering PRP at the time of injury, rib fractures underwent accelerated cellular recruitment to the fracture site, earlier soft callus formation, and increased endochondral ossification as determined by cartilage/collagen content and calcium deposition, suggesting an accelerated rate of healing. In addition, although differences in mechanical analysis did not achieve statistical significance, PRP-treated ribs trended toward increased toughness, flexural modulus, and flexural modulus compared with control ribs. Prior work has demonstrated that PRP application was associated with decreased expression of inflammatory markers, improved collagen deposition, improved neovascularization, and strengthened mechanical integrity of incorporated mesh after hernia repair.^7,8,13 In bone, it has been demonstrated that the application of growth factor–rich PRP in a spine fusion model enhanced the osteoinductive properties of the scaffold, resulting in accelerated and improved bone regeneration and remodeling.¹¹ In addition, prior work in soft tissue demonstrated comparable findings, where PRP application was associated with decreased expression of inflammatory markers, improved collagen deposition, improved neovascularization, and strengthened mechanical integrity of incorporated mesh after hernia repair.^7,8,13 Similar improvements in osteogenesis, increased mineralized bone volume on micro-CT, enhanced callus and trabecular bone formation, and improved mechanical strength have been demonstrated in long bones repair after growth factor delivery via super-concentrated platelets, although this had never been demonstrated in rib fracture repair.^18,19 The mechanism by which PRP application accelerates rib fracture healing in this model is predicted to be driven by augmented inflammatory cell, fibroblast, chondrocyte, and osteoblast localization to the fracture site, leading to earlier initiation and resolution of the inflammatory phase and subsequent transition from hematoma to soft callus with chondrocyte recruitment and improved endochondral ossification.^13,20 This is significant, as earlier achievement of a sufficiently strong callus allows for accelerated and more effective bone remodeling leading to improved chest wall function and stability, thus leading to improved pain in patients with rib fractures.²¹ Existing literature on the use of PRP on wound and fracture healing in the clinical setting has been mixed but promising. Systematic reviews have shown that the population that benefits most from PRP is that of delayed union or nonunion in long-bone fractures.^22,23 While a handful of case reports have shown the potential for PRP as an adjunct to fracture healing in a variety of other bones, such as metatarsal, patella, fibula, femur, hallux sesamoid, and scaphoid, the evidence in larger clinical outcomes studies on acute fracture healing, tibial osteotomies, and lumbar pathologies does not clearly demonstrate a beneficial effect of PRP on healing.^22,24–29 Other animal studies in rabbit, rat, and dog fractures (tibial, femur) have shown overall benefit to bone healing with the application of PRP.³⁰ In the context of these studies, our findings that PRP enhances rib fracture healing in a rat model is expected and serves as the first step in determining if PRP is able to serve as an adjunct to delayed union or nonunion rib fractures. While the preclinical findings are supportive of the use of PRP in rib fracture repair, criticisms of PRP treatment include the lack of translation to clinical studies. Platelet-rich plasma is collected autologously, and the quantity of platelets, growth factors, and leukocytes is variable, so characterization and standardization of dose should also be defined.^30,31

The potential clinical utilization of PRP for rib fracture treatment is promising. Surgical fixation has demonstrated benefit in the most severely injured; however, it is costly with nonnegligible morbidity, and its efficacy in most patients is still debated. While single growth factor delivery such as bone morphogenetic proteins have previously demonstrated potential as an adjunct in fracture repair, they are costly with short half-lives and may present a dose-dependent risk for malignancy, highlighting the need for an alternative biomimetic adjunct to accelerate rib fracture healing.^32–34 Compared with the cost of rib plating hardware and equipment, PRP preparation is extremely economical, requiring only basic phlebotomy, conical tubes, standard pipettes, and a basic centrifuge.³⁵ Although the model used in this study administered PRP directly to the open fracture site, PRP could be administered minimally invasively via ultrasound-guided percutaneous injection into the fracture site. This could be done as an alternative to surgical fixation in high-risk patients, decreasing associated procedural/perioperative costs and mitigating the postoperative and incisional pain associated with both open and thoracoscopic rib plating. By treating rib fractures via this minimally invasive method using PRP as a biomimetic medium for growth factor delivery, the potential of PRP administration for rib fracture treatment may offer a way to obviate the need for surgery while retaining the positive effects of early surgical stabilization of rib fracture.

Although the results of this study provide fodder for further investigations into the effects of PRP on rib fracture healing, there are a few limitations that must be acknowledged. For example, lack of statistical significance achieved in mechanical testing was likely secondary to a type II error because of our small sample size. Our statistical power could have been improved by increasing the number of rats tested. This study examined the effects of PRP administration on rib fracture healing but did not adequately investigate the potential pulmonary complications associated with the inadvertent, yet often inevitable, introduction of PRP in the pleural space. Rats have notoriously resilient pulmonary capacity and will often thrive with only one lung. Thus, while none of the rats had any visible pulmonary deficits on gross postmortem examination, the results seen here cannot properly address the potential associated pulmonary complications, which could occur in humans. For this, further large animal studies are necessary prior to translation of this method to the clinical trial sphere. In addition, the larger callus formed after application of PRP may increase pain by incorporating and stretching the intercostal nerve. This potential complication would benefit from further investigation. It must also be acknowledged that the experimental mechanism of rib fracture using an osteotomy created with wire cutters after dissection down to the bone differs from blunt force as the most common mechanism in traumatic rib fracture, which typically results in a less controlled fracture and potentially more bone fragments if comminuted. In addition, PRP application immediately after fracture is not clinically relevant, as most patients would receive this in the hospital hours after their injury. The reason we chose to perform dissection and a controlled osteotomy with immediate PRP application was to ensure that the fracture and delivery of PRP were exact; however, future studies will benefit from the use of larger animal models with blunt mechanisms of injury, polytrauma, and PRP application at different time points after injury, to improve clinical translation. While the findings within this study are promising, a more nuanced understanding of the cellular mechanisms underlying the observed accelerated callus maturation would also better serve our understanding of the effects of PRP on rib fracture healing. In future studies, more directed immunohistochemistry, flow cytometry, and cytokine assays at earlier time points of fracture healing can be leveraged to characterize the cellular and cytokine milieu throughout the healing process.

To conclude, rib fractures are a common traumatic injury with significant morbidity, and new approaches aimed at accelerated fracture healing could improve clinical outcomes. Growth factor delivery via super-concentrated platelets to ribs at the time of fracture increased callus size, increased cartilage and collagen deposition, and accelerated endochondral ossification as determined by calcium deposition on micro-CT leading to a trend toward improved stability. These findings indicate that PRP may be a useful adjunct to improve fracture healing in rib fracture patients at risk of pulmonary complications.

Supplementary Material

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