Softening dense tissue encourages wound repair

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By softening a cell’s nucleus so that it can squeeze its way through dense connective tissues, a group of researchers believe they’ve demonstrated a new way to help the body efficiently repair injuries.

The team of researchers from the University of Pennsylvania tested this theory by using a medication to inhibit enzymes in the nucleus of knee’s meniscus cells, which allowed the cells to move through environments that were previously impenetrable. This study was published in Science Advances.

The study focuses on cells in the meniscus, which is a thin layer of dense connective tissue in the human knee. However, the approach could prove effective beyond that specific area.

“In this case, we studied how meniscus cell nuclei can be softened to promote their migration through meniscus tissues. We have also shown similar enhancement of cell migration in other types of connective tissues, such as tendons or the cartilage covering the ends of bones,” said the study’s first author, Su Chin Heo, Ph.D., an assistant professor of research of Orthopaedic Surgery, who works within the McKay Orthopaedic Research Lab.

The paper’s corresponding author, Robert L Mauck, Ph.D., the Mary Black Ralston Professor of Orthopaedic Surgery and director of the McKay Lab, noted that “this finding may pave the way for new therapeutics to improve endogenous repair of a number of dense connective tissues that have poor natural healing capacity and are prone to failure.”

After an injury, the body requires cells to move into the afflicted area and deposit new tissues so that the tissue can be repaired, like a truck delivering cement to a construction site.

Allowing cells to move more freely into these areas could make healing quicker and/or more efficient. However, the team believed that stiff nuclei were the limiting factor, especially when it came to dense tissue such as the meniscus in the knee.

Moving through this type of tissue could rupture or otherwise damage a repair cell’s nucleus as it tried to squeeze through the tight spaces between cells. As such, damage to tissue like the meniscus could heal poorly, if at all, and result in frequent reinjury.

To remedy that, the team of researchers applied an inhibitor drug to cells called trichostatin A (TCA) that makes the proteins within their nuclei soften up, allowing for the nucleus as a whole to become more malleable.

In the truck analogy, this would be like switching from a rigid truck trailer to one with a canvas cover so that it could access a job site at the end of a road with low-hanging trees. The cover could bend as it made its way through the branches but not get hung up or damaged like a boxy, metal trailer would.

In the study, the teams saw that isolated meniscus cells that had been treated with TCA were able to move through areas that were once thought to be impassible to reach defects in tissue.

This is important becomes some of the repair methods used for injuries involve fibrous scaffolding, which can also be dense and impenetrable. These areas, too, could be infiltrated with the repair cells whose nuclei were softened, the study showed.

Moving forward, the researchers are preparing to conduct trials of this technique in meniscus tears in large animals. There is also a possibility that this work has applicability beyond just musculoskeletal injuries.

This isn’t something we’ve tested yet, but this approach could potentially be used to enhance the wound healing process of other types of tissues, such as in the skin,” Mauck said.


A fracture is a breach in the structural continuity of the bone cortex, with a degree of injury to the surrounding soft tissues. Following the fracture, secondary healing begins, which consists of four steps:

  1. Hematoma formation
  2. Fibrocartilaginous callus formation
  3. Bony callus formation
  4. Bone remodeling

Failed or delayed healing can affect up to 10% of all fractures and can be due to various factors like comminution, infection, tumor, and disrupted vascular supply. During this article, we will work through each of these steps in order and detail before then touching on primary healing, factors affecting fracture healing, and methods of stimulation of fracture healing.[1][2]

Issues of Concern
The mechanism of fracture healing is an intricate and fluent process. This process can be broken down into four stages. However, these stages have considerable overlap.

Hematoma Formation (Days 1 to 5)

This stage begins immediately following the fracture. The blood vessels supplying the bone and periosteum are ruptured during the fracture, causing a hematoma to form around the fracture site.

The hematoma clots, and forms the temporary frame for subsequent healing. The injury to bone results in the secretion of pro-inflammatory cytokines like tumor necrosis factor-alpha (TNF-α), bone morphogenetic proteins (BMPs), and interleukins (IL-1, IL-6, IL-11, IL-23).

These cytokines act to stimulate essential cellular biology at the site, attracting macrophages, monocytes, and lymphocytes. These cells act together to remove damaged, necrotic tissue and secrete cytokines like vascular endothelial growth factor (VEGF) to stimulate healing at the site.

Fibrocartilaginous Callus Formation (Days 5 to 11)

The release of VEGF leads to angiogenesis at the site, and within the hematoma, fibrin-rich granulation tissue begins to develop. Further mesenchymal stem cells are recruited to the area and begin to differentiate (driven by BMPs) to fibroblasts, chondroblasts, and osteoblasts.

As a result, chondrogenesis begins to occur, laying down a collagen-rich fibrocartilaginous network spanning the fracture ends, with a surrounding hyaline cartilage sleeve. At the same time, adjacent to the periosteal layers, a layer of woven bone is laid down by the osteoprogenitor cells.

Bony Callus Formation (Days 11 to 28)

The cartilaginous callus begins to undergo endochondral ossification. RANK-L is expressed, stimulating further differentiation of chondroblasts, chondroclasts, osteoblasts, and osteoclasts. As a result, the cartilaginous callus is resorbed and begins to calcify. Subperiosteally, woven bone continues to be laid down. The newly formed blood vessels continue to proliferate, allowing further migration of mesenchymal stem cells. At the end of this phase, a hard, calcified callus of immature bone forms.

Bone Remodelling (Day 18 onwards, lasting months-years)

With the continued migration of osteoblasts and osteoclasts, the hard callus undergoes repeated remodeling – termed ‘coupled remodeling.’ This ‘coupled remodeling’ is a balance of resorption by osteoclasts and new bone formation by osteoblasts.

The center of the callus is ultimately replaced by compact bone, while the callus edges become replaced by lamellar bone. Substantial remodeling of the vasculature occurs alongside these changes.

The process of bone remodeling lasts for many months, ultimately resulting in regeneration of the normal bone structure.[3][4][5][6]

An important point to expand on is endochondral ossification, which is the name given for the process of conversion of cartilage to bone. As described above, this occurs during the formation of bony callus, in which the newly formed collagen-rich cartilaginous callus gets replaced by immature bone.

This process is also the key to the formation of long bones in the fetus, in which the bony skeleton replaces the hyaline cartilage model. The second type of ossification also occurs in the fetus; this is intramembranous ossification; this is the process by which mesenchymal tissue (primitive connective tissue) is converted directly to the bone, which no cartilage intermediate. This process takes place in the flat bones of the skull.[7]

Clinical Significance
Primary bone healing is the reestablishment of the cortex without the formation of a callus. It occurs if a fracture is adequately “fixed” through reduction, immobilization, and rehabilitation. Secondary bone healing, as described above, occurs through the formation of a callus and subsequent remodeling.

By reducing and fixating, the clinician moves the two ends of the fracture into close apposition, which results in the minimal formation of granulation tissue and callus. ‘Cutting cones’ of osteoclasts cross the fracture site to the resorbed damaged bone, and ‘forming zones’ of osteoblasts lay down new bone.[5][8]

Reduction and fixation of fractures can be either open or closed. If treated as closed, this happens without the need to make an incision into the skin. Open refers to the need/choice to open the skin with a surgical incision.

If a fracture pattern appears stable, then the most appropriate method is closed. Options for this would be to use a cast (e.g., plaster of Paris), a brace or a splint. Open reduction tends to be the choice with unstable fractures and commonly occurs alongside internal fixation – hence the term ORIF.

Internal fixation involves the use of surgical implants to hold the two ends of the fracture closely opposed. Commonly used methods of internal fixation include plating, screws, wires, and intramedullary nails.

A final method of external fixation is also an option, and this involves the placing of pins through the skin, which are then held in place by an external ‘scaffold.’ This method tends to be used in complex fractures and can serve as a temporary option before internal fixation.[9]

Multiple factors affect fracture healing, which can broadly categorize into local and systemic categories.

Local factors:

  • Fracture characteristics – excessive movement, misalignment, extensive damage and soft tissues caught within fracture ends can lead to delayed or non-union
  • Infection – it can lead to poor healing and delayed or non-union.
  • Blood supply – reduced blood supply to the fracture site can lead to delayed or non-union.

Systemic factors (the presence of any of these factors predisposes to poor healing)

  • Advanced age
  • Obesity
  • Anemia
  • Endocrine conditions – Diabetes mellitus, Parathyroid disease, and Menopause
  • Steroid administration
  • Malnutrition
  • Smoking


Fractures have significant mortality and morbidity; therefore a multi-disciplinary approach is essential for good outcomes.[10][11][12]

There are multiple methods that the interprofessional team can utilize to promote/stimulate fracture healing, including:

Dietary supplements – calcium, protein, Vitamins C, and D
Bone stimulators – which can be electrical, electromagnetic, and ultrasound. The current effectiveness of these methods is still equivocal, and this area requires further research.
Bone graft – this involves the use of bone to help provide a scaffold to the newly forming bone. This graft can be from the patient’s body (autograft) or from a deceased donor (allograft).[13][14]

References

1.Morgan EF, De Giacomo A, Gerstenfeld LC. Overview of skeletal repair (fracture healing and its assessment). Methods Mol. Biol. 2014;1130:13-31. [PMC free article] [PubMed]

2.Einhorn TA, Gerstenfeld LC. Fracture healing: mechanisms and interventions. Nat Rev Rheumatol. 2015 Jan;11(1):45-54. [PMC free article] [PubMed]

3.Ghiasi MS, Chen J, Vaziri A, Rodriguez EK, Nazarian A. Bone fracture healing in mechanobiological modeling: A review of principles and methods. Bone Rep. 2017 Jun;6:87-100. [PMC free article] [PubMed]

4.Kostenuik P, Mirza FM. Fracture healing physiology and the quest for therapies for delayed healing and nonunion. J. Orthop. Res. 2017 Feb;35(2):213-223. [PMC free article] [PubMed]

5.Marsell R, Einhorn TA. The biology of fracture healing. Injury. 2011 Jun;42(6):551-5. [PMC free article] [PubMed]

6.Frost HM. The biology of fracture healing. An overview for clinicians. Part II. Clin. Orthop. Relat. Res. 1989 Nov;(248):294-309. [PubMed]

7.Berendsen AD, Olsen BR. Bone development. Bone. 2015 Nov;80:14-18. [PMC free article] [PubMed]

8.Al-Rashid M, Khan W, Vemulapalli K. Principles of fracture fixation in orthopaedic trauma surgery. J Perioper Pract. 2010 Mar;20(3):113-7. [PubMed]

9.Fragomen AT, Rozbruch SR. The mechanics of external fixation. HSS J. 2007 Feb;3(1):13-29. [PMC free article] [PubMed]

10.Karpouzos A, Diamantis E, Farmaki P, Savvanis S, Troupis T. Nutritional Aspects of Bone Health and Fracture Healing. J Osteoporos. 2017;2017:4218472. [PMC free article] [PubMed]

11.Cruess RL, Dumont J. Fracture healing. Can J Surg. 1975 Sep;18(5):403-13. [PubMed]

12.Bishop JA, Palanca AA, Bellino MJ, Lowenberg DW. Assessment of compromised fracture healing. J Am Acad Orthop Surg. 2012 May;20(5):273-82. [PubMed

13.Victoria G, Petrisor B, Drew B, Dick D. Bone stimulation for fracture healing: What’s all the fuss? Indian J Orthop. 2009 Apr;43(2):117-20. [PMC free article] [PubMed]

14.Marx RE. Bone and bone graft healing. Oral Maxillofac Surg Clin North Am. 2007 Nov;19(4):455-66, v. [PubMed]


More information: Su-Jin Heo et al. Nuclear softening expedites interstitial cell migration in fibrous networks and dense connective tissues, Science Advances (2020). DOI: 10.1126/sciadv.aax5083

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