Almost invisible, often ignored: periosteum, the living lace of bone





David B. BURR, PhD
Gerard M. GUILLOT, MS
Department of Anatomy and Cell Biology
Indiana University School of Medicine, Indianapolis
Indiana, USA

Almost invisible, often ignored: periosteum, the living lace of bone


by D. B. Burrand G. M. Guillot , USA



The periosteal membrane is thin and fibrous, and its deep layer is the source of cells responsible for the growth, development, modeling/remodeling, and fracture repair of our bones. It is highly vascularized and innervated by both sympathetic and pain-sensitive fibers. It arises from condensation of the general mesenchyme during fetal development, and is continuous with the Sharpey’s fibers that insert into bone and anchor it. The fibrous portion is composed of several collagen species as well as elastin. The cellular composition is diverse, including undifferentiated mesenchymal stem cells that can differentiate into fibroblasts, chondrocytes, or osteoblasts, the latter communicating extensively with the osteocytes in bone. These cells are under local control and are highly responsive to growth factors (eg, transforming growth factor beta) and several of the bone morphogenetic proteins, sex steroids (both estrogens and androgens), mineral-regulating hormones (eg, parathyroid hormone), and to other proteins associated with bone formation that are modulated through the Wnt pathway (eg, sclerostin). During fracture repair, the periosteum participates in, and provides cells for, both the intramembranous ossification that bridges and stabilizes the fracture, as well as the process of endochondral ossification and remodeling that eventually reestablishes the bone’s load bearing properties. It is a multifunctional tissue that permits our bone to adapt throughout life to changing mechanical, hormonal, and pathological circumstances.

Medicographia. 2012;34:221-227 (see French abstract on page 227)



The periosteum is a thin fibrocellular membrane that surrounds bone throughout life. Perhaps because of its relative delicacy, its structure and importance are often overlooked in deference to the more obvious processes that occur on the periosteal surface of the bone itself. Yet, these processes are regulated by the structure and cellular composition of the periosteal membrane. The cells of this thin layer contribute to the growth and development of the bone, repair of the bone following fracture, and the regulation of bone adaptation to mechanical stimuli. Without the periosteal membrane, none of these processes could occur because the cellular diversity and necessary coordination that underlies them would not exist.

Periosteal origins and the regulation of growth

The periosteum arises as a condensation of general mesenchyme that forms a perichondrial sheath around the cartilage anlage during development (Figure 1, page 222). It ends at the joint space, forming a perichondrial ring around the end of the cartilage model where the epiphyseal cartilage will develop. As the bone develops, osteoblast progenitor cells differentiate into osteoblasts in the deep layers of the periosteum, contributing to mineralization of an osseous ring around the anlage, and eventually to the enlargement of the bone diaphysis and apposition of new bone by intramembranous ossification. The periosteum is continuous with Sharpey’s fibers that insert into the bone and attach it firmly,1 although the strength and size of these connections are reduced with age.2 The periosteum is thought to grade into tendons and ligaments as they insert into bone,3 but there is still some debate about this.


Figure 1
Figure 1. Periosteal role in bone development.

A and C, Periosteum arises from a condensation of general mesenchyme, that originally forms a perichondrial sheath around the cartilage model. This sheath attaches to the metaphysis, and does not cross the developing joint space. B and D, The periosteum develops a deeper, more cellular layer (the cambium layer) and an outer fibrous layer. The cambium layer is responsible for the formation of a mineralized bone collar that surrounds the cartilage model. Sharpey’s fibers that insert into the bone are developed from and continuous with the periosteal tissue.






The entire periosteum is 70-μm to 150-μmthick in growing individuals,4 but thins with age as growth and appositional formation slow.2,5,6 It is typically thicker close to the metaphysis and thinner over the diaphysis (Figure 2). The periosteum in the adult is composed of two layers, an outer fibrous sheath of axially aligned collagen fibers that contains both fibroblasts and mesenchymal cells7 and which is composed of types I, III and VI collagen8,9 and elastin.10,11 Type III collagen is found in abundance in blood vessels, and may reflect the vascularity of periosteum, but because it cross-links rapidly may also function to reduce the extensibility of the tissue and enhance stability.12 The inner osteogenic or “cambium” layer contributes to the appositional growth of the bone throughout life, and includes mesenchymal stem cells, osteoblasts, and endothelial pericytes, the latter probably providing an additional pool of osteoprogenitor cells (Figure 2).13 It is also possible that some of the cells in the fibrous portion migrate into the cambium layer and contribute to bone formation. The osteoblasts of this layer are connected by their cellular processes to osteocytes within the bone. Some have suggested that there is an intermediate elastic layer containing capillaries,4,10 but this may disappear with maturity. Whether this constitutes another layer or not, it is true that the periosteum is highly vascularized and highly innervated14,15 by both sympathetic and sensory fibers.16

During growth, the periosteum migrates to cover new bone as it grows longitudinally. This migration involves the cellular layer as well as the outer fibrous layer.6 There is some evidence that the insertion of the periosteum into the mineralized bone by Sharpey’s fibers helps to regulate the longitudinal growth of the bone by constraining it, and that release of the periosteum allows additional growth.16-18 This was presumed to be a physical process because the periosteal membrane is highly prestressed and physically retracts and shortens by about 3 fold when incised from the bone.19 However, the tension generated by the fibrous periosteum and its insertions into bone has been shown to be insufficient for physical constraint.20 More recent evidence suggests that constraint may occur through cell-regulated mechanotransduction pathways that sense intracellular tension21 and promote the release of soluble inhibitory factors by periosteal cells.22,23 When the periosteum is released, bone morphogenetic proteins (BMPs) such as BMP-2 and BMP-4 are produced,24 which stimulates a proliferative reaction that causes growth.

Cells of the periosteum

In its different layers, the periosteum contains an entire smorgasbord of skeletal cell types at different stages of skeletal development, from mesenchymal stem cells, to chondrocytes, fibroblasts, and cells of the entire osteoblastic lineage. This accounts for its broad potential to create and shape the bone throughout growth, and for its utility as a source of cells for orthopedic procedures such as resurfacing of cartilage surfaces in degenerating joints.

The cells in the cambial layer of the periosteum are highly osteogenic, and respond to mechanical stimulation, infection, and tumors. They are highly proliferative and capable under these conditions of forming either highly organized lamellar bone, or highly disorganized woven bone in pathological situations. Because mesenchymal cells are also present, however, cells in the deep layer of the periosteum can also differentiate into chondroblasts, and form cartilage, most notably in adults during the fracture healing process. The diversity of tissue-forming potential in this area is critically important during fracture healing (see below).

Cells in the cambium layer of the periosteum express markers for both osteogenic and chondrogenic lineages. Like progenitors and fully differentiated bone and cartilage cells in other locations, these cells also are responsive to regulation by a wide range of growth factors and other proteins. Perhaps most prominently, transforming growth factor beta (TGF-β) appears to promote chondrogenic activity, but may inhibit differentiation of osteoblast progenitors.25 The cartilage-derived morphogenic proteins (CDMP) are also known to drive periosteal cells into the chondrogenic pathway,26 whereas both CDMP and parathyroid hormone–related protein (PTHrP), the latter in response to Indian hedgehog expressed by hypertrophic chondrocytes, may contribute to chondrocyte regulation during the early stages of growth, or in fracture healing.27 Bonemorphogenetic proteins,most notably BMP-2 and BMP-4, may promote the proliferation and differentiation of osteogenic cells,28 and are known to be expressed particularly during fracture healing. BMP-7 is expressed during periods of endochondral ossification, both in growth and in fracture healing.29

Although osteoclasts are blood-derived rather than bone-derived, modeling of the bone during growth requires the presence of cells that can develop into osteoclasts. Periosteum is highly vascularized, and these vessels can transport monocytes that are found in the mesenchyme of the developing limb. Type IV collagenase, which is a marker for preosteoclast development, has been immunolocalized within the deep and fibrous layers of the periosteum. Osteoclasts are thought to migrate from the more superficial layers of the periosteum, through the deeper layers to the bone surface where they can begin to shape the bone. This migration is prevented by TGF-β30 and by matrix metalloproteinases,31 suggesting again an antagonistic relationship between chondrogenic and osteogenic processes during growth.


Figure 2
Figure 2. Photomicrographs of periosteum from rat.

A, The thickness of the periosteum varies along the length of the bone, being
thicker closer to the metaphysis and B, thinner along the diaphysis. Stained with
hematoxylin and eosin.
Abbreviations: B, bone; CP, cambium layer of periosteum; FP, fibrous periosteum;
M, muscle.



Periostin is a protein important during development that, in the skeletal tissues, is localized to the periosteal membrane and to the periodontal ligament. It is an intriguing, but not yet well understood, candidate to regulate cellular processes within the periosteum, and controls the osteogenic potential of the periosteum. Periostin regulates cellular adhesion and recruitment,32 and may be either a positive33 or a negative34 regulator of osteoblast differentiation. When its promoter binds to Twist, a transcription factor important in osteogenesis and also important in determination of cell type and cell differentiation, it prevents the conversion of preosteoblasts to fully differentiated osteoblasts capable of making bone.35 Thus, upregulation of Twist and expression of periostin prevent intramembranous ossification and periosteal apposition of lamellar bone.

Periostin has a variety of isoforms, not all of which are localized to the same location or behave in like manner, making their role in the regulation of cellular differentiation and bone formation a complex matter. One of these, periostin-like factor (PLF) has been detected during embryogenesis both in mesenchymal cells in the periosteum, and in osteoblasts along trabecular bone, a location where periostin protein itself is not found.36 Moreover, PLF accelerates the differentiation of precursors into functioning osteoblasts, and promotes bone formation,37 which may be different from the action of periostin itself.34 Also, PLF is upregulated during fracture repair, whereas periostin seems to negatively regulate mineralization of the newly forming callus.sup>34 Thus, periostin likely prevents differentiation of osteoblasts and reduces bone formation, whereas its isoform PLF appears to promote differentiation and osteogenesis.

Although the number of osteogenic cells in the cambium layer declines with age,24 this seems to have little effect on its ability to respond to a mechanical stimulus. It is well known that periosteal apposition of bone continues throughout life, partially compensating for the loss of bone from other surfaces. In animal models, the significant reduction of cells in the cambium layer of the periosteum is associated with reduced chondrogenesis with age,83 but the potential to heal a fracture is not known to diminish with age in humans, in the absence of other metabolic abnormalities.

The periosteal role in fracture repair

The periosteum plays a central and multifaceted role in the processes of fracture repair. The plethora of mesenchymal stem cells can differentiate into either osteoblasts or chondroblasts under the multiple molecular signals that are released during the initial inflammatory stage of repair.39,40 The periosteum thereby participates in both the intramembranous bone formation and the processes of endochondral formation and ossification that occur during the healing process.41


Figure 3
Figure 3. Periosteal contributions to fracture healing.

A, Within hours after the fracture occurs, a hematoma develops between the fractured surfaces and within the marrow cavity. This hematoma will provide cells to reconstruct the damaged periosteum. B, Woven bone forms through intramembranous ossification and the periosteum begins to repair. The woven bone stabilizes the fracture so that pockets of cartilage can form, produced by chondroblasts that originate from mesenchymal stem cells provided by the periosteum. C, The fracture continues to heal through a process of endochondral ossification. Chondrocytes in the cartilage begin to hypertrophy, allowing calcification to begin. D, Cartilage continues to calcify, and is internally remodeled to form pockets of bone. E, The outer callus begins to remodel and reshape the bone. Cells for this process come from the vascularized periosteum and from bone-forming cells in the cambium layer. Thus, the periosteum is involved in all phases of fracture healing, providing cells for intramembranous ossification and woven bone, endochondral ossification, and finally for bone modeling and remodeling.



Bone fracture healing is commonly described to be divided into four stages: an inflammatory stage, during which a hematoma is formed and the initial molecular signals for repair are generated; periosteal woven bone formation, which bridges and stabilizes the fracture gap; cartilage formation and endochondral ossification; and finally, bone remodeling to return the bone to its original lamellar structure and external shape (Figure 3). The initial inflammatory stage incorporates a hematoma that extends into the periosteum and stimulates the proliferation of periosteal osteoprogenitors within the first two days. This may initially be under the stimulus of insulin-like growth factor I (IGF-I) and PTHrP and their receptors, which are sensitive to inflammatory mediators in the hematoma, and which are upregulated in the cambium layer of the periosteum within 24 hours following the fracture.27 By day 3, the proliferative response is at its peak, concurrent with expression of BMP- 2,3,4,5,8, and noggin within the periosteum.29 There is also an early (within 3 days) upregulation of periostin during fracture healing. Subsequently, committed progenitor cells from the periosteum migrate and differentiate to begin forming woven bone a few millimeters from the fracture site.42,43 This process eventually creates a bridge between the two ends of the broken bone, and forms a cortical collar that will later be remodeled.

Recent studies using green fluorescent protein (GFP) reporter mice have shown the sequence of events leading to this intramembranous ossification, but it is still unclear whether the cells involved are only osteoprogenitor cells or also include pericytes and dedifferentiated lining cells.43 Concurrently, or shortly after this, multipotent periosteal progenitor cells proliferate26 and migrate to the fracture site, become chondrogenic, and begin the process of cartilage formation within the fracture gap.44 By days 4-7 after the fracture, osteoblasts adjacent to the fracture site in the subperiosteal region upregulate the expression of osteocalcin and osteonectin within the zone of intramembranous ossification. These periosteal cells also produce types I and V collagen, the latter possibly contributing to the assembly and orientation of the type I collagenous matrix. At the same time, both osteonectin and osteopontin are highly expressed at the junction between the intramembranous and endochondral ossification fronts, probably signaling ossification. By day 14, periosteal osteoprogenitor cells are no longer proliferating in the area of intramembranous ossification, but the process of ossification of cartilage in the fracture gap continues. Subsequently, the calcified cartilage within the gap and the woven bone adjacent to the fracture begin to remodel and reshape the bone. Thus the periosteum is involved in all phases of the fracture repair process, providing cells for both osteogenesis and chondrogenesis, participating in both the intramembranous and endochondral ossification processes, and ultimately reshaping the bone and reestablishing normal microstructure and material properties by modeling the external shape and regulating diaphyseal curvature.

Bone modeling, remodeling, and periosteal apposition

Although the periostealmembrane thins and becomes less cellular with age, it maintains the capability for apposition of new lamellar bone throughout life. Periosteum is highly mechanosensitive, and the pluripotent osteo- and chondroprogenitor cells that reside in it are more mechanically sensitive even than mesenchymal stem cells. Periosteal apposition occurs in both men and women as they age, although the amount of apposition that occurs in women is insufficient to offset the large losses of bone from trabecular and endocortical compartments, or to maintain premenopausal bone strength.

It was an adage for years that the periosteal surface of bone is immune to bone resorption or to coupled remodeling, except perhaps during modeling processes near the bony metaphysis during growth. Although it is true that for most of adult life, periosteal bone is more osteogenic than resorptive, remodeling involving resorption does occur on this surface, particularly in older people.45 It is not difficult to find erosion cavities on the surface of the femur, for instance, in people in their 9th decade. This is likely part of the life-long adaptive process.

The periosteum is very responsive to a number of hormones, but often responds to them differently than do other bone envelopes (eg, endocortical, trabecular, and intracortical). During the period of growth and maturation, the periosteal surface of bone is particularly responsive to growth hormone (GH) and IGF-I, both of which promote appositional growth during development. However, the estrogens and androgens are also important influences on appositional growth both before and after puberty, and both are probably required for periosteal expansion. Androgens stimulate periosteal apposition in both sexes, but low levels of estrogen increase the sensitivity of androgens on the periosteal surface, even in boys.46,47 This may be the reason that aromatase-deficient boys with normal androgen levels have smaller bones. This interaction of estrogen and androgen on periosteal apposition may persist throughout life.48

It is well documented that postmenopausal estrogen deficiency is associated with periosteal apposition, and that estrogen supplementation reduces expansion,49 although it is not clear whether this is a direct effect of estrogen or a mechanical compensation for the loss of bone on the endocortical surface. However, the picture is complicated by the presence of two estrogen receptor (ER) subtypes, ER-αand ER-beta;, that may be antagonistic. Some animal experiments suggest that interaction of estradiol with ER-α promotes periosteal expansion,50 whereas ER-βinhibits periosteal apposition.50,51

Mice in which ER-α is inactive have thinner bones,52 but this is not necessarily the case in animals in which ER-β is knocked out.53 Whether this is a direct effect, or an indirect one involving coregulation with IGF-I is not clear, as ER-α knockout mice have lower IGF-I levels, whereas ER-β knockout mice have higher levels.53 This has led some to speculate that the compensatory effects of IGF-I on GH may be more important than direct effects of estrogen on periosteal osteoblasts.

The idea that ER-β is a negative regulator of periosteal apposition is consistent with the observation that apposition is suppressed in estrogen-replete women and that this inhibition is removed in estrogen deficiency. However, the picture is complicated by the fact that in humans, unlike in mice, ER-α predominates over ER-β,54 and so the antiapoptotic55 and pro-osteogenic effects of ER-α are inconsistent with the observation of the normal premenopausal suppression of periosteal apposition in women, or the postmenopausal periosteal expansion. It is possible that the two receptors interact in ways that cause different effects when only one is present,56 or that the relative importance of the receptors is gender-specific, with ER-α achieving greater effect in the male skeleton, but the presence of both being a requirement for apposition in the female skeleton.57 There may also exist a more complex relationship in which receptor signaling depends on higher or lower threshold levels of estrogen.

Likewise, the responsiveness of cells in the periosteum to IGF-I may help to explain the stimulatory effect of parathyroid hormone (PTH) on periosteal apposition.58,59 Intermittent delivery of the recombinant 1-34 fragment of human PTH (rhPTH [1-34]) is suspected to promote periosteal apposition, and its effect on bone strength has been partly explained by this phenomenon. PTH (1-34) is known to prevent apoptosis of periosteal osteoblasts,60 which could partly account for its effect on the cells in the osteogenic layer of the periosteum. PTH signaling also down regulates Sost expression in osteocytes,61 which has been shown in mice with a constitutively active PTH receptor to increase periosteal bone formation. Sclerostin, the protein which the Sost gene encodes, down regulates bone formation through the Wnt pathway. The inhibition of Sost expression in osteocytes by PTH increases bone formation on the periosteal surface. This demonstrates that the osteogenic cells in the periosteum are in communication with cortical osteocytes, which help to regulate periosteal bone formation through a Wnt-dependent pathway.

Conclusion

The periosteal membrane provides cells for growth, development, maturation, adaptation, and repair of our bones throughout our entire life. It is an important target tissue that maintains our skeletal health and well-being by adapting to our changing developmental, hormonal, and mechanical needs over the many decades of our life. Although often overlooked, it is vital to our skeletal health.

The authors wish to thank Dr. Keith Condon for his help in taking and editing photomicrographs in Figure 2.



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Keywords: aging; development; estrogen; fracture repair; periosteum; remodeling